JP4482267B2 - Conjugated stabilized polypeptide composition - Google Patents

Abstract

A stabilized conjugated peptide complex comprising a peptide conjugatively coupled to a polymer including lipophilic and hydrophilic moieties, wherein the peptide may be insulin, calcitonin, or an enzyme. In a particular aspect, the invention comprises an insulin composition suitable for parenteral, as well as non-parenteral administration, comprising insulin covalently coupled with a polymer including a linear polyalkylene glycol moiety and a lipophilic moiety, wherein the insulin, the linear polyalkylene glycol moiety and the lipophilic moiety are conformationally arranged in relation to one another such that the insulin in the composition has an enhanced in vivo resistance to enzymatic degradation, relative to insulin alone. The conjugates of the invention are usefully employed in therapeutic as well as non-therapeutic, or diagnostic, applications as shown in the figure.

Description

（式中、ｗ＋ｘ＋ｙ＋ｚ＝２０であり、Ｒはオレイン酸：（ＣＨ₃（ＣＨ₂）₇ＣＨ＝ＣＨ（ＣＨ₂）₇Ｃ（Ｏ）−である。）
接合体２は以下の式で表される。
【化２０】

接合体３は以下の式で表される。
【化２１】

【００７０】
接合体１は、重合体系の中心に市販のポリソルベート・モノオレート、即ち、多くの薬剤の分野で使用される糖誘導体を特徴とする。親油性および吸収性増進特性がオレイン酸鎖により付与される一方、ポリエチレングリコール（ＰＥＧ）残基は、親水性（水素結合促進）環境を提供する。インシュリンは、重合体のＰＥＧ領域に隣接するカーボネート連結体を通じて付着される。
【００７１】
接合体２において、糖残基は除かれるが、インシュリンは再度、重合体の親水性ＰＥＧ領域の隣接するカーボネート結合体を通じてポリマーに付着される。重合体の親油性の脂肪酸領域は、インシュリンへの付着点からある程度離れた位置にある。
【００７２】
結合体２に関して上述した構成は、結合体３の場合、逆となる。この場合も糖残基は除去されるが、この構造体において、親油性の脂肪酸残基はインシュリンへの付着点に最も近い位置にあり、親和性ＰＥＧ領域は同様にカーボネート結合体を通じての付着点から離れた位置にある。
【００７３】
ペプチドに対する重合体の付着点に関して親水性領域および親油性領域を各種の整合状態にすることが、本発明の広範囲の実施にて可能であり、かかる変更により、インシュリンの親油性および親水性領域を均衡性よく保護する重合体が得られる。接合体１、２および３において、重合体間のカーボネート結合体の付着点はＧｌｙ^A1のアミン官能基であることが好ましい。上述のように、ペプチドの各分子の１つに複数変位の重合体を付着させることが可能である。例えば、第２の重合体をインシュリンに付着させる場合、その付着点は、Ｐｈｅ^B1のアミン官能基を通ることが好ましい。少なくとも理論上、第３の重合体をＬｙｓ^B29のアミン官能基に付着させることが可能である。しかしながら、インシュリン分子当り２つの重合体を付着させることが妥当な誘導化の最大の限度であることが経験的に確認されている。
【００７４】
本発明の全体的な実施に当り、重合体をペプチドに結合する各種の方法が利用可能であり、これについては、以下に更に詳細に説明する。タンパク質およびポリペプチドを取り扱う場合、ペプチド内の特定の残基基は、その全体的な生物学的完全性の点が重要であることを理解すべきである。かかる残基を不当に妨害しない適当な結合剤を選択することが重要である。場合によっては、結合を阻止し、従って、こうした残基の活性を遮断することが困難であるが、付与された有利な特性を保ちつつ、安定性を増すことの犠牲としてある種の活性は失ってもよい。例えば、生体内への適用例において、投与頻度を少なくして、コストを削減し且つ患者の便宜性を増すことができる。
【００７５】
本発明に従い、タンパク質／ペプチド結合体に利用される重合体は、所望の目的を達成することを可能にする良好な物理的特性を含み得るように設定される。吸収促進剤は、細胞薄膜を通じてペプチドの浸透を可能にする一方、ペプチドの安定性は改善せず、生体内への適用例において、かかる浸透促進剤を使用しないフォーミュレーションにおいて本発明の重合体−ペプチド接合体を利用することができる。このため、本発明の１つの形態は、浸透促進剤を最小にすべく重合体内に脂肪酸誘導体を含めることに関する。
【００７６】
本発明の共有結合による重合体−ペプチド接合体において、ペプチドは不安定な化学的結合により水溶性重合体に共有結合させて付着させることができる。ペプチドおよび重合体のこの共有結合は、化学的または酵素反応により、分解させることができる。この重合体−ペプチド重合体は、許容可能な程度の活性を保持する。重合体がペプチドから完全に分解されたときに、成分としてのペプチドの完全な活性が実現される。これと同時に、接合重合体にポリエチレングリコールの一部が存在して、重合体−ペプチドに対して高度の水溶性および長期に亘る血液循環機能を付与する。糖脂質は、通常、重合体と関係付けられ、その脂肪酸成分がペプチドの疎水性領域を占め、これにより凝集を阻止する。ペプチドが凝集すると、小腸での吸収が不十分となる。凝集しないペプチドは、小腸により、より容易に吸収される。このため、糖脂質を結合重合体に含めることは、安定性を増しかつ接合後のペプチドの凝集を阻止する働きをする。上述の変性は、ペプチドに対して改良された溶融性、安定性および薄膜との親和特性を付与する。こうした改良にかかる特性の結果、本発明は、活性な重合体−ペプチド双方の非経口投与および経口投与を可能にし、また、生体への適用例において、加水分解による分解後に、ペプチドそれ自体の生体利用性を実現する。
【００７７】
以下に説明する実施例に使用される重合体は、ポリエチレングリコール変性による糖脂質およびポリエチレングリコール変性による脂肪酸として種類分けすることができる。好適な接合重合体の内、モノパルミテート、ジパルミテート、トリパルミテート、モノローレート、ジローレート、トリアローレート、モノオレート、ジオレート、トリオレート、モノスチレート、ジスチレートおよびトリスチレートを含むポリソルベートを掲げることができる。各組み合わせから得られる重合体の平均分子量は、約５００乃至約１０，０００ダルトンの範囲であることが好ましい。この実施例に好適な代替的な重合体は、ポリエチレングリコール・エーテルまたはエステルであり、かかる脂肪酸は、ローリック、パルミティックおよびステアリック酸であり、重合体の平均分子量は、５００乃至１０，０００ダルトンの範囲である。重合体には、誘導体基を有することが好ましく、この場合、かかる基は、最後がポリエチレングリコールとなり、または脂肪酸となるようにすることができる。この誘導基は、重合体内に配置することもでき、このように、ペプチドと重合体との間のスペーサー基として機能することができる。
【００７８】
所望の重合体を得るために脂肪酸・ソルビタンを変性させる幾つかの方法について、分子構造体を参照しつつ以下に更に詳細に説明する。ポリソルベートは、ソルビトールのエステルおよびその無水物であり、ソルビトールおよびソルビトール無水物の各分子毎に約２０個のエチレン酸化物の分子で共重合される。以下に、典型的な重合体の構造を示す。
【００７９】
【化２２】

【００８０】
ｗ、ｘ、ｙ、ｚの合計は２０であり、Ｒ₁、Ｒ₂およびＲ₃は各々、ローリック、オレイック、パルミティックおよびステアリン酸基からなる基から独立的に選択され、またはＲ₁、Ｒ₂は各々ヒドロキシルである一方、Ｒ₃はローリック、パルミティック、オレイックまたはステアリン酸基である。これら重合体は市販されており、薬剤のフォーミュレーションとして使用されている。高分子量の重合体が所望である場合、その重合体は、ソルビタン・モノローレート、ソルビタン・モノオレート、ソルビタン・モノパルミテートまたはソルビタン・モノステアレートのような糖脂質および適当なポリエチレングリコールから合成することができる。開始試薬として使用することのできる糖脂質の構造体について以下に説明する。
【００８１】
【化２３】

【００８２】
３つの位置にて、ポリエチレングリコールで置換した糖脂質重合体を構成するとき、端末に２つのフリーのヒドロキシルを有する所望のポリエチレングリコールをトリチル・クロライドの１モルを使用して、ポリジン内のトリチル基にて１つの端末にて保護する。ポリエチレングリコールの残りのフリーのヒドロキシル基は、トシレートまたは臭化物に置換する。所望の糖脂質は適当な不活性溶剤中で溶融させかつナトリウム複合体により処理する。保護されたポリエチレングリコールのトシレートまたは臭化物は不活性溶剤中で溶融させ、過剰な量にて糖脂質の溶液に添加する。この生成物は、室温にて無水物の不活性溶液内でパラトルエンスルホン酸の溶液で処理し、カラムクロマトグラフで精製する。この変換の構造体について以下に説明する。
【００８３】
【化２４】

【００８４】
試薬のモル等量を調整し、かつポリエチレングリコールの適当な分子量範囲を使用することにより、上記の方法により、所望の範囲の分子量を有するモノまたは二置換性の糖脂質を形成することができる。
【００８５】
【化２５】

【００８６】
ここで、ｎおよびｍは各々独立的に変更可能であり、安定化される特定のペプチドに適した値、例えば、１乃至１６の何れかとすることができる。
【００８７】
上述の糖脂質の糖部分は、以下の構造分子式を有するグリセロールまたはアミノグリセロールと、置換することができる。
【００８８】
【化２６】

【００８９】
この変性において、一次アルコールを最初に、ローリック、オレイック、パルミティックまたはステアリックのような脂肪酸成分でエーテル化またはエステル化し、アミノ基は脂肪酸で誘導して、以下に示すようなアミドまたは二次的アミノ基を形成する。
【００９０】
【化２７】

【００９１】
ここで、ｍは例えば１０乃至１６のような任意の適当な値とすることができる。
残りの第１のアルコール基は、トリチル基により保護される一方、第２のアルコール基は、ポリエチレングリコールにより所望の重合体が変換される。
【００９２】
通常、ポリエチレングリコールは、一端に分離する基を有し、その他端にメタオキシ基を有する。ポリエチレングリコールは不活性溶剤中で溶融させ、糖脂質およびナトリウム複合体を含む溶液に添加する。
【００９３】
この生成物は、以下に示すような所望の重合体が得られるように、パラトルエンスルホン酸内で室温にて保護を解除する。
【００９４】
【化２８】

【００９５】
例えば、ポリソルベート・トリオレートのような脂肪酸の２つまたは３つの分子で置換されたポリソルベートと同様の特定の物理化学的性質が得られるよう、ポリエチレングリコール鎖の別の部分に脂肪酸誘導体を含めることが望ましい場合がある。
【００９６】
この重合体を示す構造体は、以下の反応式にてポリソルベートの開放鎖として示してある。
【００９７】
【化２９】

【００９８】
【化３０】

【００９９】
重合体Ａを合成するとき、例えば、ソルケタールのようなグリセロールの第１および第２の炭素にてヒドロキシル成分を保護することが望ましい。残りのヒドロキシル基は不活性溶剤中でナトリウム塩に変換し、ハロゲン化またはトシル化したポリエチレングリコールと反応させポリエチレングリコールの一端からエステルとして保護されるようにする。このグリセロール保護を解除し、形成される２つの自由ヒドロキシル基を対応するナトリウム塩に変換する。これらの塩は、脂肪酸で一部誘導したポリエチレングリコールにて不活性溶剤中で反応させる。自由ヒドロキシルがトシルまたは臭素に変換された後に反応が行われる。
【０１００】
重合体Ｇは、同一の方法で合成されるが、酸の端末炭素にてハロゲン化された脂肪酸のエステルと保護されたグリセロールとを最初に反応させる。
【０１０１】
重合体Ｃを合成するとき、１、３−ジハロ−２−プロパノールで開始することが望ましい。このジハロ化合物は不活性溶剤で溶融させ、１モルの脂肪酸成分で予め誘導された２モルのポリエチレングリコールのナトリウム塩で処理する。この生成物は、クロマトグラフ、または透析で精製する。形成される乾燥生成物は、不活性溶剤中でナトリウム水素化物で処理する。このようにして形成されたナトリウム塩は、部分的に保護されたポリエチレングリコールのハロ誘導体と反応させる。
【０１０２】
重合体の糖部分を省くことが望ましい場合がある。形成される重合体は、依然としてポリエチレングリコール成分を含む。脂肪酸成分の薄膜親和特性は、固有の脂肪酸を親脂性の長繊維アルキンで置換することにより保持することができ、このため生体適合性が保たれる。この実施例の一例において、２つの端末自由ヒドロキシル基を有するポリエチレングリコールを不活性溶剤中でナトリウム水素化物で処理する。脂肪酸状成分の第１の臭素誘導体の１つの等価重量をポリエチレングリコール溶剤の混合体に添加する。所望の生成物は、不活性溶剤中で抽出し、必要であれば、カラム・クロマトグラフにより精製する。
【０１０３】
【化３１】

【０１０４】
ここで、脂肪酸とポリエチレングリコールとの間でエステル連結体を形成することが望ましい場合、この酸の酸塩化物は、適当な不活性溶剤中で余剰な所望のポリエチレングリコールで処理する。重合体は、不活性溶剤内で抽出され、必要であればクロマトグラフにより更に精製する。
【０１０５】
【化３２】

【０１０６】
ある種のペプチドの変性例において、脂肪酸成分をペプチドに直接、接合することが望ましいことがある。この場合、重合体は、脂肪酸分子に置かれた誘導可能な官能基により合成する。不活性溶剤中の適当な分子量のモノメタオキシル・ポリエチレングリコールの溶液は、ナトリウム水素化物で処理し、その後、酸の端末炭素に分離基を有する脂肪酸のエチルエステルを含む溶液を添加する。この生成物は、溶剤抽出後に精製し、必要であれば、カラム・クロマトグラフにより精製する。
【０１０７】
【化３３】

【０１０８】
希釈酸または塩基で処理することにより、エステルの保護を解除する。
【０１０９】
【化３４】

【０１１０】
ポリペプチドとのカーボネート結合体を形成することが望まれる場合、当該技術分野で公知の化学的還元法により、カルボキシルまたはエステルをヒドロキシル基に変換する。
【０１１１】
【化３５】

【０１１２】
ポリペプチド接合体に使用される官能基は、通常、重合体の末端にあるが、場合によっては、その官能基が重合体内に位置することが好ましいことがある。この状況のとき、誘導基がスペーサとして機能する。この実施例の一例において、脂肪酸成分は、炭素αにてカルボキシル基に臭素化して、酸成分は、エステル化することができる。かかる型式の化合物に対する実験方法は、上述の方法と同様であり、以下に示した生成物が得られる。
【０１１３】
【化３６】

【０１１４】
長いスペーサが望ましいとき、ポリエチレングリコール・モノエーテルをアミノ基に変換し、脂肪酸成分により誘導された無水ケイ酸で処理することができる。所望のポリエチレングリコールを支持する一次アミンは、ｐＨ８．８のナトリウム・リン緩衝液に溶融させ、以下の等式に示すように、置換した無水ケイ酸の脂肪酸成分で処理する。この生成物は、溶剤抽出により分離し、必要であれば、カラム・クロマトグラフにより精製する。
【０１１５】
【化３７】

【０１１６】
上述の反応過程は、説明の便宜のためにのみ掲げたものであり、本発明の広い範囲の実施に当り、ペプチドを変性するのに有利に使用することのできる、例えば、溶融性、安定性、非経口投与および経口投与のための細胞膜との親和性を達成するために利用することのできる反応剤および構造体に限定するものであると解釈されるべきではない。
【０１１７】
本発明は、例えばペプチド、タンパク質、酵素、成長ホルモン、成長因子等のような生物的な活性な長分子、診断用試薬との生体的に適合可能な重合体の結合体を提供するものである。かかる長分子化合物は、１つのアミド連結体内で接合したアルファ・アミノ酸で形成して、ペプチド・オリゴノマーおよび重合体を形成することができる。これら物質の機能に依存して、ペプチド成分は、タンパク質、酵素、成長ホルモン等とすることができる。明確化のため、これらの物質は、全体として以下にペプチドと称し、Ｐｒで表示する。全ての場合、生物学的に活性なペプチドは、フリーのアミノまたはカルボキシル基を含む重合体とペプチドとの結合は、一般に、フリーのアミノ、またはカルボキシル基を通じて行われる。
【０１１８】
本発明の説明のために選択したペプチドは、医薬、農業、科学および家庭用並びに工業用の分野で特に有益である。これらのペプチドは、置換治療で利用される酵素、動物の成長促進ホルモン、細胞培養における細胞成長ホルモン、または、例えば、バイオテクノジーおよび生物学的および医療用診断のような各種の分野で使用される活性タンパク質性物質とすることができる。酵素としては、スーパーオキシドジムスターゼ、インターフェロン、アスパラギナーゼ、アルギナーゼ、アルギニンデアミナーゼ、アデノシンデアミナーゼ、リボヌクレアーゼ、トリプシン、キモトリプシン、パパインがあり、ペプチドホルモンとしては、インシュリン、カルシトニン、ＡＣＴＨ、グルカゴン、ソマトスタチン、ソマトトロピン、ソマトメジン、副甲状腺ホルモン、エリスロポエチン、視床下部放出因子、プロラクチン、甲状腺剌激ホルモン、エンドルフィン、エンケファリン、バソプレシンがある。
【０１１９】
共有結合した生成物を得るための重合体とペプチドとの反応は、容易に行われる。説明の簡略化のため、重合体は（Ｐ）で表示する。重合体がヒドロキシル基を含む場合、重合体は最初にパラ・ニトロフェルカルボネートのような活性なカルボネート誘導体に変換される。この活性な誘導体は、次に、緩やかな条件下で短時間にてペプチドと反応して、生物学的活性を保つカルバミン酸エステルの誘導体を発生する。
【０１２０】
【化３８】

【０１２１】
上述の反応および試薬は、単に説明のためのものであり、限定的なものではない。ウレタン、またはその他の結合体を生じるその他の活性試薬を採用することもできる。ヒドロキシル基は、当該技術分野で公知の試薬を使用して、アミノ基に変換することもできる。そのカルボキシル基を通じてその後にペプチドと結合する結果、アミドが形成される。
【０１２２】
重合体がカルボキシル基を含む場合、該重合体は、混合した無水化物に変換し、ペプチドのアミノ基と反応させ、アミド結合体を含む接合体を形成することができる。その他の方法において、カルボキシル基は、水溶性のカルボジイミドで処理し、ペプチドと反応させ、アミド結合体を含む接合体を生じさせることができる。
【０１２３】
ペプチド結合体の活性および安定性は、重合体とペプチドとの分子量比率を変更し且つ異なる分子寸法の重合体を使用する等の各種の方法で変更することが可能である。接合体の溶融性は、重合体組成物に含まれるポリエチレングリコール成分の比率および寸法を変えることで変更することができる。親水性および親油性の特性は、脂肪酸成分とポリエチレングリコール成分とを注意深く組み合わせることにより、均衡させることが可能である。
【０１２４】
以下には、本発明の重合体をペプチド接合体との変性反応の幾つかの例が示してある。
【０１２５】
【化３９】

【０１２６】
Ｉ、ＪおよびＫを含む上記の反応過程において、接合重合体の親水性／親脂性の釣り合わせを変性する手順が明らかにされている。接合重合体内のエステル基は、エステラーゼによる加水分解を受け易く、このため、エステル基を含む接合重合体は、エステル基を加水分解に対する抵抗性の大きいエーテル基に変換することができる。ＬおよびＭを含む反応過程は、ヒドロキシル基をカルボキシル基に変換する状態を示す。この点に関し、カルボキシル基は、ヒドロキシルよりも安定化機能に優れた（イオン調和させた複合体を形成する）カルボキシレート・アニオンを提供し、このアニオンは、かかる複合体からは形成されない。本発明の重合体を含む調和したイオン複合体を形成するためのその他の適当なアニオン源の官能基は、硫酸およびリン酸基を含む。
【０１２７】
一般に本発明の重合体−ペプチド接合体の安定特性を向上させるためには、各種の技術を有利に採用することができる。これら技術には、次のものが含まれる。即ち、例えばエステル基をその他の基に変換する上述した例のような重合体を加水分解に対する抵抗性に優れた基で官能基とすること、重合体により安定化されたペプチドに適するように接合重合体の親脂性／親水性の釣り合いを変性すること、および重合体により安定化されるペプチドの分子量に対し重合体の分子量を適正なレベルに調整することである。
【０１２８】
本発明を治療目的に適用するために価値のある、ポリアクリレート・グリコール誘導による重合体のユニークな特性は、全体として生体適合性、つまり生理的適合性があることである。重合体は、各種の水溶性の性質を有し、また毒性がない。これら重合体は、非抗原性であり、また、非免疫原性で、酵素の生物学的活性を妨害しない。これら重合体は、血液内で長期に亘り循環し、生体器官から容易に排泄される。
【０１２９】
本発明の生成物は、ペプチドの生物学的活性を保持するのに有用であることが、確認されており、例えば、水または許容可能な液体媒体中で溶融させることにより、例えば、治療の投与のために調合することもできる。非経口投与または経口投与により投与される。非経口投与のために微細なコロイド状懸濁液を調合し、滞留効果を提供し、または経口により投与することができる。
【０１３０】
凍結乾燥状態において、本発明のペプチド−重合体の接合体は、貯蔵安定性に優れ、また、本発明の接合体の溶液フォーミュレーションも同様に貯蔵安定性に優れることを特徴とする。
【０１３１】
本発明の治療用重合体−ペプチド接合体は、そのペプチドの成分が有効であるあらゆる症状または病状の予防または治療のために利用することができる。
【０１３２】
更に、本発明の重合体−ペプチド接合体は、生物系あるいは種の成分、状態または病状の診断および非生理系統の診断の目的に利用することが可能である。
【０１３３】
更に、本発明の重合体−ペプチド接合体は、植物系の予防または症状あるいは病状の治療に適用することが可能である。一例として、接合体のペプチド成分は、各種の植物系に使用可能であり、殺虫性、除草剤、殺菌剤および／または殺虫剤効果を有する。
【０１３４】
更に、本発明の接合体のペプチド成分は、抗体とし、または診断、免疫検定および／または分析目的のために抗原の特性を持つようにしてもよい。
【０１３５】
治療目的に使用するとき、本発明はかかる症状または病状があり、またはかかる症状または病状に罹る可能性があり、かかる治療を必要とする動物の治療方法に適用することも可能であり、上記症状または病状の治療に有効である本発明の重合体−ペプチド接合体の有効量をその動物に投与する段階を含む。
【０１３６】
本発明の重合体−ペプチド接合体により治療すべき対象は、人間および人間以外の動物（例えば、鳥、犬、猫、牛、馬）の双方を含み、哺乳類であることが好ましく、更に、人間であることが最も好ましい。
【０１３７】
対処すべき特定の症状または病状に依存して、動物には、本発明の重合体−ペプチド接合体を任意の適当な治療上有効な投与量で投与し、この投与量は当業者の技術の範囲内で、また、不必要な実験を行わずに、容易に判断することができる。
【０１３８】
一般に、治療効果を達成するために化学式１の化合物の適当な投与量は、１日当り、受手の体重１ｋｇあたり１μｇ乃至１００ｍｇの範囲であり、１日当り、体重の１ｋｇ当り１０μｇ乃至５００ｍｇの範囲であることが好ましく、また、１日当り、体重１ｋｇ当り１０μｇ乃至５０ｍｇの範囲であることが最も好ましい。この所望の投与量は、１日の適当な間隔にて２回、３回、４回、５回、６回またはそれ以上に分けて投与することが好ましい。こうした少量ずつの投与量は、単位投与量の形態にて投与することができ、例えば、１０μｇ乃至１０００ｍｇの範囲とし、単位投与量の形態に当り、活性成分の５０μｇ乃至５００ｍｇの範囲であることが好ましく、また、５０μｇ乃至２５０ｍｇの範囲内であることが最も好ましい。これと代替的に、受手の状態が必要とするならば、投与量は連続的な注入として投与することもできる、もちろん、投与方法および投与形態は、所定の治療目的に望ましく且つ有効である化合物の治療量に影響する。
【０１３９】
例えば、経口的に投与するときの投与量は、同一の活性成分を非経口投与するときの量の少なくとも２倍とし、典型的に例えば２乃至１０倍とする。
【０１４０】
本発明の重合体−ペプチド接合体は、それ自体を投与し、および薬学的に許容可能なエステル、塩およびその他の生理的に有効な誘導体の形態として投与することが可能である。また、本発明は獣および人間の治療の双方に適する薬剤フォーミュレーションを対象としており、このフォーミュレーションは、活性剤として本発明の１または複数の重合体−ペプチド接合体を含む。
【０１４１】
かかる薬学的および医療用フォーミュレーションにおいて、活性剤は、薬学的に許容可能な１または複数の担体と共に、および選択随意により、その他の治療成分と共に利用される。該担体は、フォーミュレーションのその他の成分と適合し、その受手に不当に害にならない点で薬学的に許容可能なものでなければならない。活性剤は、上述のように所望の薬理的効果を達成する量にてかつ所望の１日当りの投与を達成するのに適した量にて投与する。
【０１４２】
このフォーミュレーションは、非経口投与およびその他の投与法に適したものを含んでおり、具体的な投与方法には、経口、直腸、頬側、局所的、鼻、眼、皮下、筋間、静脈、皮膚、鞘内、関節内、心房内、くも膜下、気管支、リンパ管鞘および子宮内投与がある。経口投与および非経口投与に適したフオーミュレーションであることが好ましい。
【０１４３】
液体溶液を含むフォーミュレーションに活性剤を利用するとき、そのフォーミュレーションは、経口または非経口投与することが有利である。活性剤が液体懸濁フォーミュレーションに採用され、または生体適合性担体フォーミュレーションの粉末として採用される場合、そのフォーミュレーションは、経口、直腸または気管支を通じて投与することが有利である。
【０１４４】
活性剤が粉末固体の形態にて直接利用される場合、その活性剤は経口投与することが有利である。これと代替的に気管支に対して、担体ガス中に粉末を噴霧して粉末をガス状に分散させ、患者が適当なネブライザー装置を備える呼吸回路からそのガスを吸引するように投与してもよい。
【０１４５】
本発明の活性剤を含むフォーミュレーションは、単位投与量の形態にて便宜に提供することができ、また、製薬分野で周知の任意の方法により調合してもよい。かかる方法は、一般に、活性化合物を１または複数の付随の成分を構成する担体と関係付ける段階を含む。典型的にフォーミュレーションは活性化合物を液体担体、微細に粉砕した固体担体、或いはその双方と均一に且つ密接に接触させることにより調合され、次に必要であれば、その製品の形状を所望のフォーミュレーション投与の形態にする。
【０１４６】
経口投与に適した本発明のフォーミュレーションは、カプセル、カシュ剤、錠剤またはトローチのような別個の単位として提供することができ、その各々が粉末または粒子または水溶液、またはシロップ、エリキシル剤、エマルジョン、またはドーナツのような水以外の液体中の懸濁液として活性成分の所定の量を含む。
【０１４７】
錠剤は圧縮または成形により、選択随意により１または複数の補助的な添加物を加えることにより形成することもできる。圧縮した錠剤は、適当な機械で圧縮し、活性化合物が粉末または粒子のように自由に流動する形態とし、それを選択随意にバインダ、崩壊剤、平滑剤、不活性希釈剤、表面活性剤または放出剤と混合させる。粉末状の活性化合物と適当な担体の混合物からなる成形錠剤は、適当な機械で成形することにより製造することができる。
【０１４８】
例えば、スクロースのような糖の濃縮水溶液に活性化合物を添加し、任意の補助的な成分を添加することにより、シロップを製造することができる。かかる補助的な成分は、風味剤、適当な防腐剤、糖結晶化の遅延剤、および、例えばグリセロールまたはソルビトールといったポリヒドロキシル・アルコールのようなその他の成分の溶融性促進剤を含むこともできる。
【０１４９】
非経口投与に適したフォーミュレーションは、活性化合物の無菌水溶液調合剤を調合する段階を含み、これは受手の血液に対し等張性であることが望ましい（例えば、生理的な食塩溶液）。かかるフォーミュレーションは、懸濁剤および濃縮剤、または血液成分、または１または複数の器官に対して化合物を標的決めし得るようにしたその他の微粒子系を含むことができる。このようなフォーミュレーションは、単位投与量または多数投与量の形態で提供することもできる。
【０１５０】
鼻噴霧機フォーミュレーションは、防腐剤および等張性剤にして活性化合物を精製した水溶液を含む。かかるフォーミュレーションは鼻の粘膜に適合したｐＨおよび等張性の状態に調整することが好ましい。
【０１５１】
腸投与のためのフォーミュレーションは、ココアバター、水和化油脂または水和化油脂カルボキシル酸のような適当な担体と共に、座薬として提供することができる。
【０１５２】
眼炎用フォーミュレーションは、ｐＨおよび等長性ファクタを眼の状態に適合するよう調整することが望ましい点を除いて、鼻噴霧の同様の方法で調合することができる。
【０１５３】
局所用フォーミュレーションは、鉱物油、石油、ポリヒドロキシル・アルコールまたは局所用薬剤フォーミュレーションに使用されるその他の基のような１または複数の媒体中に溶融しまたは懸濁させた活性化合物を含む。
【０１５４】
上述の成分に加えて、本発明のフォーミュレーションは、希釈剤、緩衝剤、風味剤、崩壊剤、表面活性剤、濃縮剤、平滑剤、防腐剤（酸化防腐剤を含む）等から選択した１または複数の補助的成分を更に含むことができる。
【０１５５】
本発明の治療以外の適用例において、重合体−ペプチド接合体は、ペプチドと重合体成分との共有結合、またはそれと代替的に、非共有的な結合関係を利用することができる。更に、本発明の共有結合した重合体−ペプチド接合体の説明に関係して以下に説明するような適当な投与方法により、治療用ペプチド剤を投与するときに関係付けられたペプチドおよび重合体成分を利用することができる。
【０１５６】
かかる治療以外の適用例において、関係付けられたペプチド−重合体成分、即ち、ペプチドおよび重合体成分は、最初に相互に調合して、安定性および劣化抵抗性を向上させることができる。これと代替的に、これらの成分は、例えば、多数成分の別個の成分とし、その成分を使用時点で混合し、その成分が形成される混合体中で重合体とペプチドとが関係可能に結合しないとき、迅速に劣化しまたはその他の劣化作用を受けるようにする。関係したペプチドおよび重合体組成物の形態に関係なく、本発明はかかる関係可能な重合体が存在しないとき、ペプチドの何らかの特性または形態を改善し、或いは、ペプチドの成分に関してペプチドの効果を向上させる関係を対象とする。
【０１５７】
従って、本発明は治療以外の適用例の好適な一例として、溶液内のペプチドを生体外で安定化させる適当な重合体を提供することを目的とする。例えば、重合体は、ペプチドの熱安定性を増し且つ酵素による劣化抵抗性を増すために採用することができる。本発明の方法による共有結合を介してペプチドの熱安定特性を促進する結果、貯蔵寿命、室温の安定性、診断および研究用試薬、および例えば、免疫検定キットのようなキットの堅牢さを改善する手段が得られる。具体的な一例として、本発明に従い、アルカリフォスファターゼを適当な重合体に共有結合し、または関係可能に結合させ、生物流体中の抗体または抗原を比色検出するキットの試薬として使用するとき、かかるフォスファターゼに安定性を付与することもできる。
【０１５８】
【実施例】
以下に、本発明を実施例を挙げてさらに詳細に説明する。以下の実施例は、本発明を限定するものではない。
【０１５９】
［実験例Ｉ］
［接合体１］
ポリソルベート・トリオレート・ｐ−ニトロフェニル・カーボネート ５０ｍｌの無水アセトニトリル中のｐ−ニトロフェニルクロロフォルメート溶液（０．８ｇ、４モル）に乾燥ポリソルベート・トリオレート（７ｇ、４モル）を添加し、次に、ジメチルラミノピリジン（０．５ｇ、４モル）を添加する。この反応混合体は、室温で２４時間攪拌する。低圧下にて溶剤を除去し、形成される析出物は、乾燥ベンゼンで希釈し、セルーテを通じてろ過する。残留物は、乾燥ベンゼン中で一昼夜、冷蔵し、更なる析出物は、ろ過により除去する。溶剤は低圧下にて除去し、残留ベンゼンは低圧にて排出することにより除去し、６．４ｇのポリソルベート・トリオレー卜ｐ−ニトロフェニル・カーボネートが得られるようにする。
【０１６０】
インシュリンと活性重合体との結合 蒸留水中の活性ポリソルベート・トリオレートｐ−ニトロフェニル・クロロフォルメート（１ｇ）の溶液に０．１Ｍｐ、Ｈ８．８のリン酸塩緩衝液中のウシインシュリン溶液（５０ｍｇ）を添加する。必要に応じて１Ｎ・ＮａＯＨを添加することにより、ｐＨを保つ。反応混合体は、室温で２．５時間攪拌する。この時点で、混合体はセファデックスＧ−７５を使用して、ゲルろ過クロマトグラフィを行う。０．１Ｍｐ、Ｈ７．０のリン酸塩緩衝液により、溶離させて精製し、自動成分採取器により成分を採取すれば、接合体１が得られる。この重合体の成分は、トリニトロベンゼン・スルホン酸（ＴＮＢＳ）分析により重合体の成分を測定し、また、ビューレット法によりタンパク質の濃度を測定する。重合体とインシュリンとのモル比率は、１対１と測定する。
【０１６１】
［実験例II］
［接合体２］
ポリエチレングリコール・モノスチアレートの端末ヒドロキシル基を上述のように、ｐ−ニトロフェニル・クロロフォルメートと反応させることにより、活性化させる。蒸留水中の活性重合体（１ｇ）の溶液に、ｐＨ８．８にて０．１ＭｐＨリン酸塩緩衝液中に溶融させたウシインシュリン（８０ｍｇ）を添加する。
【０１６２】
このｐＨは、１Ｎ・ＮａＯＨにより慎重に調整することにより維持する。３時間の攪拌後、反応混合体は余剰なグリシンで急冷して、セファデックスＧ−７５を使用して、ゲルろ過クロマトグラフィを行う。インシュリン−重合体の接合体を採取し、凍結乾燥させた。タンパク質成分は、ビューレット分析法により測定し、定量値を求める。
【０１６３】
［実験例III］
［接合体３］
テトラヒドロ−２−（12-bromododecanoxy）−２Ｈピロン ピリジニウムｐ−トルエンスルホン酸塩（Ｐ−ＴＳＡ）を含むジクロロメタン中の１２−ブロモ−１−ドデカノール（１モル）の溶液に、ジヒドロピラン（２モル）を添加する。反応混合体は、２４時間攪拌し、次に水で２回洗浄し、無水ＭｇＳＯ4により乾燥させる。ジクロロメタンを低圧下で除去する。必要であれば、形成される生成物は、シリカゲルによりクロマトグラフィにより精製する。
【０１６４】
ポリエチレングリコールとテトラヒドロピラン誘導体との結合 乾燥ベンゼン中に溶融した上述のテトラヒドロピラン誘導体をＮａＨ（１モル）を含む乾燥ベンゼン中のポリエチレングリコール（１モル）の溶液に添加する。反応混合体は、室温にて２４時間攪拌する。その時間後、混合体はベンゼンによりシリカゲルカラムを通じて溶離させる。必要であれば、カラムクロマトグラフィにより更に精製する。保護テトラヒドロピラン基を室温にてｐ−ＴＳＡで処理することにより除去する。必要であれば、カラムクロマトグラフィにより最終生成物を精製する。重合体のヒドロキシル基は、上述のように、ｐ−ニトロフェニル・クロロフォルメートとの反応により活性化させる。インシュリンとの接合は、接合体１に関して説明したように行う。
【０１６５】
［実験例IV］
重合体−インシュリン接合体、および自然のインシュリンについてウシインシュリンを使用して比較試験を行い、動物におけるその相対的安定性およびその活性を判断した。動物について研究した場合、重合体−インシュリンの血圧降下の効果を自然のインシュリンの効果と比較した。平均体重２５ｇの雌および雄の白子鼠を一昼夜断食させ、２日間に亘り各種の段階で行った各治療に対し、５匹を１つの群として使用した。
【０１６６】
各試験動物による自然のインシュリン（群１、１００μｇ／ｋｇ、皮下注射）、自然のインシュリン（群２、１．５ｍｇ／ｋｇ、ガバージュ）。
【０１６７】
接合体１） 群３、１００μｇ／ｋｇ、経口投与）、または接合体１（群４、１００μｇ／ｋｇ、皮下注射）を１回、時間零の時点で投与した。追加の群（群５）には、何れの種類のインシュリンも投与せず、所定の採取時間の３０分前にグリコース３０の負荷を加えた。動物は治療前、一昼夜および研究期間中、断食させた。
【０１６８】
試験材料は、リン酸塩緩衝食塩水ｐＨ７．４内で調合した。インシュリンで治療した後、０．５、１、２、４、８および２４時間の所定の採取時間の３０分前に、動物には大量のグルコースの負荷を加えた（５０％溶液として５ｇ／ｋｇ、経口投与）。各動物にインシュリン、または接合体１の１回の投与量、および１回のグルコース負荷量のみが投与されるようにした。所定の採取時間にて、血液を尾血管から採取し、ワンタッチ・デジタル・グルコース・メータ（ライフ・スキャン）を使用して、血糖値を直ちに分析した。その試験結果は、群１−５について、図１に掲げてある。
【０１６９】
群１の動物（自然のインシュリン、皮下注射）の血糖値は、３０分の試験時点にて、対照（群５、非処理）動物の約３０％であった。この低血糖効果は、群１の動物にて僅か３．５時間しか継続しなかった。経口投与した自然のインシュリン（群２）は、血糖値を対照の最大６０％まで低下させ、この最大の応答性は、インシュリンで治療した後、３０分の時点で生じる。一方、群３の動物（接合体１、１００μｇ／ｋｇ、ｐ．ｏ．）における血糖値は、低血糖活性の見かけの遅延した開始に伴い低下した。群３の動物の低血糖活性は、群３に投与されたインシュリンの投与量が群２に投与された僅か１／１５にも拘らず、群２の動物よりも顕著であった。３時間後の全ての時点にて、群３の動物の血糖値は、その他の治療群よりも低く、４時間乃至８時間の採取時点にて最大の差が生じる。群４の動物（接合体１、１００μｇ／ｋｇ、ｓ．ｃ．）の血糖値は、研究の最初の４時間、群１の動物の血糖値と同一の経過であった。４時間後、群２の血糖値は、対照（非処理、群５）の値よりも上廻る一方、８時間にて、群４の血糖値は群５の血糖値の６２％にて以下し、群５の血糖値よりも低い状態を保った。
【０１７０】
［実験例Ｖ］
非接合形態のインシュリンおよび接合体１を試験材料として使用して、雄および雌白子鼠についてインシュリンの効果実験を行った。この実験の１つの目的は、接合体１の形態によるインシュリンを皮下注射したときに、インシュリンと同様の方法で血糖値に対する効果があるか否かを判断することである。第２の目的は、自由インシュリンと異なり、接合体１のインシュリン複合体が経口投与したとき、血液の血糖値を低下させる作用があるかどうかを判断することである。その結果は図２に掲げてあり、この場合「インシュリン複合体」は、接合体１を意味する。
【０１７１】
断食し未治療の１０匹の白子鼠（雄、雌５匹ずつ）から採取した基準血液検体を採取して、血漿グルコース分析を行った。図２に基準値は、符号「Ｏ」で示してある。３つの追加の群（それぞれ雄、雌５匹ずつ）一昼夜断食させ、ガバーシュによる経口投与でのみグルコースを投与した（体重１ｋｇ当り５ｇ）。投与後、３０分、６０分および１２０分の時点にて、３回、１０匹の動物から血液検体を採取してグルコースの分析を行った。断食した鼠群（雄、雌５匹ずつ、３回の時点の各々で殺して血液分析される）に対して経口投与（ｐ．ｏ．）および非経口（ｓ．ｃ．）の双方で、各投与毎に市販のインシュリンおよび接合体１を投与し、異なる治療群が得られるようにした。この治療、投与経路および図２の実験結果について示した符号は次の通りである。
【０１７２】
（i）グルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「●」、（ii）インシュリン（１００μｇ／ｋｇ、ｓ．ｃ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「▽」、（iii）インシュリン（１．５ｍｇ／ｋｇ、ｐ．ｏ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「▽」、（iv）接合体１（１００μｇ／ｋｇ、ｓ．ｃ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「□」、（ｖ）接合体１（２５０μｇ／ｋｇ、ｓ．ｃ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「□」、（vi）接合体（１．５ｍｇ／ｋｇ、ｓ．ｃ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「△」。接合体１のこうした試験において、投与した溶液中のタンパク質の濃度は、０．１ｍｇタンパク質／ｍｌ溶液であり、比較の目的のため、０．７８ｍｇタンパク質／ｍｌ溶液の投与した溶液中のタンパク質濃度である変性共有結合によるインシュリン−重合体接合体が含まれる。（vii）変性結合体１（１００ｐｇ／ｋｇ、ｓ．ｃ．）およびグルコース（５ｇ／ｋｇ、ｐ．ｏ．）、記号「△」。
【０１７３】
インシュリンは、グルコースの負荷を加える前の１５分の時点で投与した。グルコースは、５ｇ／ｋｇの投与量にて、基準動物群を除く全ての群に、ガバージュにより経口投与した（標準的な食塩水中の５０％ｗ／ｖ溶液の１０ｍｇ／ｋｇ）。インシュリンをガバージュにより経口投与したとき、その投与量は１．５ｍｇ／ｋｇとした（標準的食塩水中の０．００８％ｗ／ｖ溶液の１８．８５ｍｌ／ｋｇ）。インシュリンを皮下注射で投与したときその投与量は１００μｇ／ｋｇとした（標準的食塩水中の０．００４％ｗ／ｖ溶液の２．５μｌ／ｋｇ）。
【０１７４】
接合体１重合体−インシュリン複合体をｇａｖａｇｅにより経口投与したとき、その投与量は１．５６ｍｇ／ｋｇ（非希釈試験材料の２．０ｍｌ／ｋｇ）の投与量で投与した。接合体１の重合体−インシュリン複合体を皮下注射により投与したとき、その投与量は、１００μｇ／ｋｇ（投与した０．７８ｍｇ／ｍｌ溶液を１対１０で希釈した１．２８μｌ／ｋｇ）。または２５０μｇ／ｋｇ（投与した溶液の１対１０で希釈した分の３．２０ｍｌ／ｋｇ）。変性した接合対１は、０．１ｍｌ／ｋｇのインシュリン／ｍｌを含み、１．０ｍｌ／ｋｇの量にて投与し、１００μｇ／ｋｇの投与量とした。
【０１７５】
ジェミニ遠心力分析、および市販のグルコース試薬キットを使用して、グルコースを測定した。この分析と共に、ｈｅｘｏｋｉｎａｓｅにより触媒作用を加えたＡＴＰとグルコースとの反応に基づき、酵素分析を行い、また、グルコース−６−リン酸塩脱水反応を行い、ＮＡＯＨを得た。同様の検体を分析し、その平均値を求めた。一部の血漿検体は、特定の検体における極めて高いグルコース濃度を判断するため、希釈（１対２または１対４）が必要であった。
【０１７６】
グルコースの負荷を加えた後、３０分にて平均血漿グルコース値は、高濃度となり、６０分にて低下し、１２０分にて基準値以下となった。市販のインシュリンを皮下注射で投与した場合（体重１ｋｇ当り１００μｇ。これは、血糖値の上昇を防止するのに極めて効果的であった）。しかしながら、インシュリンを経口投与したとき（１．５ｍｇ／ｋｇの多量の投与量にて）、血糖値の上昇に対する効果は全くなかった。インシュリン、タンパク質は、消化経路内で容易に加水分解され、血流中に完全な状態で吸収されないから、このことは予想された。
【０１７７】
接合体１を１００または２５０μｇ／ｋｇの投与量の何れかにて皮下注射したとき、該接合体はグルコースの負荷を加えた後、血糖値の上昇を制限するのに極めて効果的であった。３０分および６０分双方の時点にて、接合体１を１００μｇ／ｋｇ投与した後、１００μｇ／ｋｇの自由インシュリンを投与した場合よりも平均血漿血糖値は著しく低下した。接合体１を２５０μｇ／ｋｇ投与したときの平均血漿血糖値は低く、３０分の時点で著しくはないが、６０分の時点では著しく低く、１２０分の時点で基準値に戻った。自由インシュリンを１００μｇ／ｋｇ、および接合体１を１００μｇ／ｋｇの双方共に投与したとき、１２０分の時点での血糖値は基準値以下であった。
【０１７８】
変性接合体１を１００μｇ／ｋｇの投与量で投与したとき、３０分の時点で血糖値は著しく低下した。
【０１７９】
［実験例VI］
［ポリソルベート・モノパルミテートのパラーニトロフェニル・カルボネートの調合］
最初に乾燥ベンゼンを使用する共沸により、ポリソルベート・モノパルミテートを乾燥させる。
【０１８０】
１０ｍｌの乾燥ピリジン内の乾燥重合体（２ｇ、２モル）の溶液に、パラ−ニトロフェニルクロロフォルメート（０．６ｇ、３モル）を添加する。この混合体を室温にて２４時間、攪拌する。反応混合体は、氷で冷却し、乾燥ベンゼンで希釈して、フィルタを使用してろ過する。この手順を反復し、最終的に、回転蒸発器にて溶剤を除去する。残留する溶剤は、真空吸引器で除去する。生成物の量は１．８ｇである。
【０１８１】
［実験例VII］
［インシュリンとのポリソルベート・モノパルミテート接合体の調合］
上述した実験例１の接合反応手順に従い、１ｇの量のポリソルベート・モノパルミテートおよび８０ｍｇのインシュリンを使用し、反応生成物をＨＰＬＣで分離すると、インシュリン−ポリソルベート・モノパルミテートの共有結合した接合体が得られる。
【０１８２】
［実験例VIII］
［酵素−重合体接合体の調合］
実験例１の接合体１に関して説明した方法と同一の方法を使用して、アルカリフォスファターゼ（ＡＰ）を重合体に結合させた、更に、重合体対タンパク質の比率を高くするかまたは低くする何れかが有利かを判断するため、１４０モルの重合体／酸素モルと、１４モルの重合体／酵素モルを使用して接合体を調合した。接合体ＡＰの分子当りの重合体群の数は、重合体の高および低比率に対し、それぞれ３０および５である。
【０１８３】
アルカリフォスファターゼの約５群／分子を得るために、次の手順を採用した。０．０５Ｍナトリウム・バイカーボネートに４．１ｍｇ（無塩分）を溶融させた。水／ジメチル−スルホキシド内でこの溶液に活性重合体（０．７５ｍｇ）を添加し、その溶液を室温にて３乃至１２時間、攪拌した。形成された反応混合体を、透析管（ＭＷカットオフ１２，０００−１４，０００）内で１２時間に亘り塩溶液（０．３Ｎ・ＮａＣｌ）に対して透析し、透析溶液は４乃至６回交換した。高比率に対して同一の手順を行った。透析材料の総タンパク質濃度は、ビューレット法により測定した。
【０１８４】
［活性の測定および安定性の試験］
Ａ．ボーラー（ｖｏｌｌｅｒ）その他の者の文献ＷＨＯ、５３、５５（１９７６）方法に従い、フォスファターゼ分析を行った。マイクロウェルのアリコット（５０μｌ）を添加し、２００μｌの基礎溶液（２０％のエタノラミン緩衝液内の４−ニトロフェニルリン酸塩、１０ｇ／ｌ、ｐＨ９．３）と混合させ、室温にて４５時間、保温した。５０μｌの３Ｍ・ＮａＯＨにより反応を停止させた。この吸収率は、マイクロプレート読取り装置内で４０５ｎｍにて測定した。
【０１８５】
フォスファターゼの活性を各種の条件下で天然の酵素と比較した。同様の濃度のアルカリフォスファターゼおよびアルカリフォスファターゼ−重合体接合体を含む希釈溶液を各種の温度で貯蔵した。酵素の活性を定期的に試験した。５℃、１５℃、３５℃および５５℃で試験した２種類の重合体を５℃で貯蔵した対照アルカリ・リン酸塩と比較した。
【０１８６】
表Ａから理解されるように、双方の重合体の最初の酵素活性は、対照剤よりも約３倍大きい値であった。双方の重合体−酵素接合体は、自然の酵素よりも熱安定性が優れていて、これは重合体対酵素の比率が高いことを特徴とする接合体の場合、特に顕著である。
【０１８７】
【表１】

【０１８８】
［実験例IX］
［接合体１］
Ａ．ｐＨ９．２の０．０５Ｍナトリウム・バイカーボネート緩衝液中のインシュリン溶液（５０ｍｇ）に水−ジメチルスルホキシド中の活性重合体溶液（１ｇ）を添加し、室温にて３時間、攪拌した。この混合体のｐＨは、１Ｎ・ＮａＯＨにより慎重に調整することにより維持する。次に、この反応混合体を０．１Ｍ−ｐＨ７．０のリン酸塩緩衝液で透析する。精製された生成物を凍結乾燥する。ビューレット検定法により、タンパク質含有量（４８ｍｇ）を測定する。インシュリンに結合された重合体鎖の数は、ＴＮＢＳ検定により求め、重合対２モル対インシュリン１モルの比率となるようにする。
【０２００】
［実験例XVI］
次の方法にて、糖尿病（ＢＢ）鼠モデルにて、重合体−インシュリンを評価する。ＢＢ鼠は、人間のインシュリン依存性糖尿病の信頼性の高いモデルである。
【０２０１】
ＢＢ鼠は、毎日、経皮的（ｓｃ）にインシュリン投与を行わないと、２日以内で死亡する。１４日間の試験中、ｓｃインシュリンを停止し、動物を、低（１００μｇ／ｋｇ／日）、中（３ｍｇ／ｋｇ／日）、高（６ｍｇ／ｋｇ／日）の量の経口投与による重合体−インシュリンの治療に切り替えた。その結果（平均生存時間）が図６に掲げてある。ｓｃから経口投与に急激に切り替えることは、重合体インシュリンの特に苛酷な試験である。その結果から、低投与量の動物は、生存するために十分なインシュリンが受けられず、中程度の投与量の群は、生存時間が多少伸び、高投与量群の一部の動物は、経口投与した重合体インシュリンの投与を受ける試験の期間中（１４日間）、生存したことが分かる。重合体インシュリンは、好適でないフォーミュレーションにてガバージュ投与した。また、この試験の結果から、ａｍ血糖値と比べてｐｍが著しく低下し、また、１回大量に投与するのではなく、１日に何回も分けて投与した場合、動物を一層良くコントロールし得ることが分かった。重合体−インシュリンを正常な（糖尿病でない）動物に毎日投与する結果、ａｍ血糖値が著しく低下する。
【０２０５】
本発明を実施する最良の形態 本発明の現在の好適な接合体安定化によるポリペプチド重合体組成物およびフォーミュレーションは、共有結合したペプチド複合体に関し、ここでペプチドはその一体部分として例えば、線状ポリアルキレングリコールのような親水性成分を含む重合体の１または複数の分子に共有結合され、また、該重合体は、その一体部分として親油性成分を含む。
【０２０６】
本発明の特に好適な形態は、重合体と共有結合した生理的に活性なペプチドを含む生理的に活性なペプチド組成物に関し、該重合体は次のものを含む。即ち、（Ｉ）線状ポリアルキレングリコール成分と、（II）親油性成分とである。ここで、ペプチド、線状ポリアルキレングリコール成分および親油性成分は、互いに関して調和状態に配置され、生理的に活性なペプチド組成分中の生理的に活性なペプチドは、生理的に活性なペプチド単独の場合と比較して（即ち、結合した重合体が存在しない接合体の形態のとき）、酵素による劣化に対する体内抵抗性が増大している。
【０２０７】
更に、好適な形態において、本発明は、次のものを含むポリソルベート複合体と共有結合した生理的に活性なペプチドからなる３次元的に調和した生理的に活性なペプチド組成物に関する。即ち、（ｉ）線状ポリアルキレングリコール成分と（ii）親油性成分とであり、ここで生理的な活性なペプチドは、線形ポリアルキレングリコール成分および親油性成分は、互いに関して調和可能に配置され、親油性成分が３次元的に調和した状態で外部から利用可能であり、また、（ｂ）生理的に活性なペプチド組成物中の生理的に活性なペプチドは、生理的に活性なペプチド単独と比べて酵素による劣化に対する体内抵抗性が増大している。
【０２０８】
更に別の好適な形態において、本発明はトリグリセリド本体成分と炭素原子にて結合されたポリアルキレングリコールスペーサ基を通じてトリグリセリド本体成分に共有結合された生物学的に活性なペプチドを有するトリグリセリド本体成分からなるマルチリガンド接合ペプチド複合体に関し、該複合体は、トリグリセリド本体成分の炭素原子に直接共有結合されるか、またポリアルキレングリコールスペーサ成分を通じて共有結合された少なくとも１つの脂肪酸成分からなっている。かかるマルチリガンド接合ペプチド複合体において、トリグリセリド生物学的活性成分のαおよびβ炭素原子は直接共有結合させて付着され、またはポリアルキレングリコールスペーサ成分を通じて間接的に共有結合された脂肪酸成分を含むことができる。これとは別に、脂肪酸成分は直接、またはポリアルキレングリコールスペーサ成分を通じてトリグリセリド本体成分のα炭素に共有結合することができ、生物学的に活性なペプチドは直接共有結合させ、またはポリアルキレンスペーサ成分を通じて間接的に共有結合されてトリグリセリド成分のβ炭素に共有結合される。
【０２０９】
本発明の産業上の利用可能性 本発明は本明細書に開示された接合安定ペプチド組成分を薬剤の経口投与およびペプチドは不十分な病状の治療に使用することを目的とするものである。
【図面の簡単な説明】
【図１】図１は、インシュリン自体および複合体の形態にて投与するため、分を単位とする時間を関数として、血漿グルコース量をｍｇ／ｄＬの単位で示すグラフである。
【図２】図２は、インシュリンを各種の形態にて投与する時間（時間）を関数とする血漿グルコース量のｍｇ／ｄＬ単位で示す図である。
【図３】図３は、時間を関数とするインシュリンおよびＯＴインシュリンのキモトリプシン消化のグラフである。
【図４】図４は、鼠にカルシトニン接合体を経口投与したときの時間を関数とする鼠における血漿カルシウム量のグラフを示す図である。
【図５】図５は、鼠に重合体カルシトニンＯＴ１−ｃｔまたはＯＴ２−ｃｔ重合体カルシトニン接合体を経口投与したときの時間を関数とする鼠のセラム・カルシウム量を示す棒グラフを示す図である。
【図６】図６は、糖尿病の鼠における重合体−インシュリンの効果の棒グラフを示す図である。
【図７】図７は、シノモルグス猿における００１−インシュリンを経口投与した効果を経過時間に亘り血液グルコース中の濃度変化として示すグラフを示す図である。
【図８】図８は、シノモルグス猿における００１−インシュリンを経口投与したときの経過時間に亘る投与効果を示す棒グラフを示す図である。[0001]
The present invention relates to the field of conjugate stabilizing polypeptide composition inventions. More particularly, the present invention relates to conjugate-stabilized polypeptide composition (poly) peptide and protein compositions and formulations, and methods for making and using the same.
[0002]
[Description of related technology]
The use of polypeptides and proteins for the systematic cure of specific diseases is now common in the medical field. The role that peptides play in replacement therapy is very important, and therefore many studies have been conducted with the aim of synthesizing them in large quantities by DNA recombination techniques. Many of these polypeptides are endogenous molecules that have the potential to exert their biological effects and are specific.
[0003]
One major cause of hindering the utility of using such substances for their intended application purposes is that they are easily metabolized by proteolytic enzyme plasma when administered parenterally. In addition to proteolysis within the stomach, oral administration of these substances also causes the stomach to be so acidic that it is destroyed before it reaches the intended target tissue. Therefore, there are many problems. Polypeptides and protein substances generated by the action of the stomach and proteolytic enzymes are degraded by external and internal peptidases in the intestinal brush border membrane to generate dipeptides and tripeptides, and pancreatic enzymatic proteins Even if degradation is prevented, the polypeptide is degraded by brush border peptidases. Any given peptide that survives through the stomach is further metabolized within the intestinal mucosa where the invading diaphragm prevents entry into the cell.
[0004]
Despite these obstacles, there is considerable evidence in the literature suggesting that nutrients and drug proteins are absorbed through the intestinal mucosa. On the other hand, nutrients and drug (poly) peptides are absorbed by specific peptide carriers within the intestinal mucosal cells. These findings indicate that properly formulated (poly) peptides and proteins can be administered orally and retain sufficient biological activity for their intended purpose. However, in order to ensure that its physical activity is fully retained, the peptide can be denatured to at least a significant extent, while at the same time being stable against proteolytic enzymes and its entry through the intestinal mucosa. If the functions can be improved, they can be used appropriately for their intended purpose. The product thus obtained is more efficiently absorbed, and at the same time has the advantage that a smaller dose is required for an optimal therapeutic effect.
[0005]
Problems associated with oral or parenteral administration of proteins are well known in the pharmaceutical industry, and various strategies have been adopted to solve these problems. These strategies include the addition of protein promoters such as salicylate, lipid-bail salt mixed micelles, glycerides and acrylic carnitine, but these methods involve local inflammation and toxicity, epithelial Often causes problems with significant local toxicity, such as complete wear of the layers and inflammation of the tissue. The reason for these problems is that the accelerator is usually administered with the peptide product and the dosage is often leaked. Other strategies to improve oral administration include mixing peptides with proteases such as aprotin, soy trypsin inhibitor and amalstatin to limit degradation of the administered drug. Unfortunately, such proteases are not selective and endogenous proteins are inhibited. This effect is undesirable.
[0006]
In addition, it has been studied to promote invasion of peptides in a thin film such as a mucous membrane by modifying the physicochemical properties of the target drug. Research results show that enhancing lipophilicity alone is not sufficient to facilitate cell-to-cell transport. In fact, it has been suggested that breaking the peptide hydration bond is an energy barrier that must be overcome when the peptide diffuses in the thin film (see Non-Patent Document 1). Protein stabilization has also been published by several authors. Non-Patent Document 2 discloses various methods for modifying an enzyme in order to provide a stabilized product in a water-soluble and non-immune living body.
[0007]
Numerous studies on protein stabilization have been published. Various methods for joining an enzyme and a polymerized material (for example, see Non-Patent Document 2) are disclosed. More specifically, these polymers are dextran, polyvinyl pyrrolidone, glycopeptides, polyethylene glycols and polyamino acids. The conjugated polypeptide formed is reported to retain its biological activity and remain water soluble for parenteral administration. Also, the same person discloses that polyethylene glycol makes a protein soluble and non-immune when bound to such protein (see Patent Document 1). However, such polymeric materials do not contain ingredients suitable for binding to the intestinal mucosa and do not contain ingredients that promote or enhance penetration into the membrane. Such conjugates are water soluble but are not intended for oral administration.
[0008]
It is also taught that proteins can be stabilized by conjugation with chondroitin sulfate (see Patent Document 2). The product of this combination is usually a polyanion, is very hydrophilic and lacks cell penetration. These products are usually not intended for oral administration.
[0009]
It is taught that certain acidic polysaccharides such as pectin, argesic acid, hyaluronic acid, carrageenin are combined with proteins to form both soluble and insoluble products (see, for example, US Pat. ). Such polysaccharides are poly anions obtained from edible plants. These substances lack cell penetration and are generally not intended for oral administration.
[0010]
It has been disclosed that polyethylene glycol can bind to proteins such as peroxidative disproportionating enzyme (“SOD”) (see, for example, Non-Patent Document 3). The formed conjugation protein is more stable against denaturation and enzymatic digestion. The polymer does not contain a component required for interaction with the thin film, and therefore has the same problem as described above that it is not suitable for oral administration.
[0011]
Non-patent document 4 and non-patent document in which peptides and protein drugs in which a relatively low molecular weight compound such as aminoretin, fatty acid, vitamin B12, glycoside and a protein drug substance are conjugated are stabilized. 5, Non-Patent Document 6, Non-Patent Document 7). The modifying compound is not a polymer and therefore does not contain the components needed to obtain both the solubility and the affinity for thin films required for biological action after oral and parenteral administration. Many of these studies cannot be administered to the body orally.
[0012]
Another solution for prolonging the action time of protein-based substances in vivo is sealing technology. It is taught to seal a protein drug in an azo polymer for oral administration (see Non-Patent Document 8). This film has been reported to survive digestion in the stomach, be degraded by microorganisms in the large intestine, and release the sealed protein. This technique utilizes a physiological mixture and does not promote absorption of proteins released in the thin film. Further, it is taught that physiologically active compounds including proteins can be sealed with a film by coacervation, and the final product is suitable for transmucosal administration (see Patent Document 4). Other formulations of the present invention can be administered by inhalation, oral, parenteral and transdermal administration. Such a solution does not provide complete stability against acids and gastrointestinal protein-based enzymes, which are desirable properties for oral administration.
[0013]
Another strategy for stabilizing protein drugs for oral and parenteral administration involves incorporating therapeutic agents into liposomes (see Non-Patent Document 9). The liposome-protein complex is a physiological mixture. Its administration produces irregular and unpredictable results. When using such liposomal protein complexes, it has been reported that protein components undesirably accumulate in certain organs. In addition to these factors, there are other disadvantages associated with the use of liposomes such as cost, difficult manufacturing methods that require complex lyophilization processes, and solvent incompatibility. Furthermore, when developing a clinically useful liposome formulation, the problem arises that the distribution in the living body and antigenicity change as limiting factors.
[0014]
The use of “proteinoids” has recently been announced (see Non-Patent Document 10). It has been reported that several types of therapeutic drugs are administered orally using this system, which seals the drug of interest within a polymer sheath consisting of highly branched amino acids. . As with liposomes, the drug is not chemically bound to the protinoid sphere, and the drug may leak from the compound in the dosage form.
[0015]
The peptide that is the subject of research that seeks to synthesize and to promote its administration and assimilation into the body is insulin.
[0016]
The use of insulin for the treatment of diabetes dates back to 1992, when active extracts extracted from the pancreas were shown to have therapeutic effects in diabetic dogs (see Non-Patent Document 11). Treatment of diabetic patients of the same year with pancreatic extract resulted in dramatic life-sustaining clinical improvements. For long-term recovery, a daily injection process of insulin is required.
[0017]
The insulin molecule consists of two amino acid chains linked by a disulphide conjugate. The molecular weight of insulin is about 6,000. Pancreatic islets beta cells secrete a single-chain precursor of insulin known as proinsulin. Proinsulin proteolis results in the removal of the four basic amino acids (No. 31, 32, 64, 65 in the proinsulin chain, Arg1, Arg1, Lys, Arg, respectively) and connection ("C") The peptide was removed. In the two chain insulin molecules that are formed, the A chain has glycine at the amino terminus and the B chain has the amino acid phenylalanine.
[0018]
Insulin can exist as monomers (dimers), dimers or hexamers (hexamers) formed from three dimers (dimers). Hexamer has two Zn ²⁺ Coordinate to atoms. Monomers have biological activity. Until recently, bovine and porcine insulin has been used exclusively for the treatment of diabetes in humans, but it is known that there are a number of differences in insulin by type.
[0019]
Porcine insulin is almost similar to human insulin, except that it has an alanine rather than a threonine residue at the B chain C end. Despite these differences, most mammalian insulin has specific activities that can be compared. Until recently, animal extracts have provided all the insulin used to treat this disease. With the advent of genetic engineering technology, it has become possible to produce human insulin on a commercial scale (eg, commercially available from Eli Lilly and Company, Indianapolis, IN). Humulin (Humulin® insulin).
[0020]
Insulin has been used for more than 70 years as a treatment for diabetes, but there are few studies on the stability of its formulation and only two publications have been recently published (Non-Patent Document 12, Non-patent document 13). In these publications, the authors detail the chemical stability of some insulin formulations under various temperature and pH conditions. Early reports cover almost only biological capacity as a means of stabilizing insulin formulations. However, with the advent of several new and effective analytical techniques, disk electrophoresis, size exclusion chromatography and HPLC, it is possible to examine in detail the chemical stability characteristics of insulin. Early chemical studies on insulin stability were difficult because the purity of the recrystallized insulin being examined was 80-90% or less. More recently, single-component, high-purity insulin has become available. This single component insulin contains a concentration of impurities that cannot be detected by current analytical techniques.
[0021]
Formulated insulin is prone to many types of degradation. When the side chain amino group from a glutaminol or asphalazinyl residue is hydrolyzed to the free carboxylic acid, an enzymatic deamidation is performed. There are six possible sites for such amidolysis in insulin. That is, Gln ^A5 , Gln ^A15 , Asn ^A18 , Asn ^A21 , Asn ^B3 And Gln ^B4 It is. A published report reports that three Asn residues are most susceptible to such reactions.
[0022]
Insulin in the acidic state is Asn ^A21 Has been reported to deteriorate rapidly due to remarkable amide decomposition (see Non-Patent Document 13). On the other hand, in a neutral formulation, deamidation does not depend on the insulin concentration and the source of insulin. ^B3 At a much slower rate. However, temperature and formulation type play an important role in determining the rate of hydrolysis in B3. For example, if insulin is crystalline rather than amorphous, hydrolysis at B3 is minimal. Of course, if the flexibility in the crystalline form is reduced (third structure), the reaction rate becomes slower. If the third structure is stabilized by incorporating phenol into the neutral formulation, the rate of deamidation is reduced.
[0023]
In addition to the products degraded by hydrolysis in the insulin formulation, high molecular weight modified products are also formed. Blanger et al. Have shown that the product formed when the insulin formulation is stored in the temperature range of 4 ° C. to 45 ° C. by size exclusion chromatography is a covalently bound insulin dimer. In addition, in a protamine-containing formulation, a covalently bound insulin protamine product is also formed. The formulation rate of insulin dimer and insulin protamine products is significantly affected by temperature. In the case of human or porcine insulin (regular N1 formulation), the time to formulate a 1% high molecular weight product is reduced from 154 months to 1.7 months at 37 ° C compared to 4 ° C. In the case of a porcine insulin zinc suspension formulation, the same modification takes 357 months at 4 ° C, but only 0.6 months at 37 ° C.
[0024]
This type of deterioration in insulin is very significant for the study of diabetes. The formation of high molecular weight products is generally slower than the rate at which the hydrolysis (chemical) degradation products described above are formed, but the significance is more critical. There is significant evidence that the immunological responsiveness to insulin is due to the presence of covalently bound aggregates of insulin (see Non-Patent Document 14, Non-Patent Document 15, and Non-Patent Document 16). ).
[0025]
It has been shown that 30% of diabetic patients receiving insulin administration have specific antibodies to covalent insulin dimers. Even at concentrations as low as 2%, the presence of covalently bound insulin dimers has been reported to be extremely responsive to allergic patients in lymphocyte stimulation. When the dimer content is 0.3 to 0.6%, the response is not remarkable. As a result, it is recommended that the concentration of covalent insulin dimer present in the formulation should be kept below 1% to avoid clinical symptoms.
[0026]
Several insulin formulations are commercially available. In any formulation, the stability has been improved to the extent that refrigeration is no longer necessary, but insulin formulation with improved stability remains a challenge. The development of modified insulin that does not tend to form high molecular weight products is a significant advance in the pharmaceutical and medical fields, and this (in addition to enabling oral administration of insulin) If it can be modified for stability, it will contribute significantly to the management of diabetes.
[0027]
In addition to the in vivo use of polypeptides and proteins as therapeutic agents, there is a significant increase in the use of polypeptides and proteins in the field of diagnostic reagents. In many such applications, polypeptides and proteins are used in solution, where the polypeptides and proteins are susceptible to thermal and enzymatic degradation of (poly) peptides and proteins such as: Viral proteins used in numerous analytical methods for diagnosis or testing of diseases such as enzymes, peptides and protein hormones, antibodies, enzyme protein conjugates used in immunoassays, antibody hapten conjugates, AIDS Hepatitis, rubella, eg, peptides and protein growth agents used in tissue culture, enzymes used in clinical chemistry, insoluble enzymes as used in the food industry. As a more specific example, alkaline phosphatase enzyme is widely used as a reagent for kits used to detect the presence of antibodies or antigens in biological fluids by chromaticity measurement. Such enzymes are commercially available in various forms, including free enzymes and antibody conjugates, but their storage stability and dissolution are often limited. As a result, alkaline phosphatase enzyme conjugates are often freeze-dried, and additives such as bovine serum albumin and Tween 20 are used to increase the stability of enzyme formulations. Such a solution is beneficial in some cases to increase its resistance to degradation of polypeptide and protein drugs, but has significant drawbacks that hinder its general application.
[0028]
[Patent Document 1]
US Pat. No. 4,179,337
[Patent Document 2]
US Pat. No. 4,585,754
[Patent Document 3]
US Pat. No. 4,003,792
[Patent Document 4]
US Pat. No. 4,963,367
[Non-Patent Document 1]
Conradi, R.D. A. Hilger, A. R. , HO, N.M. F. HL. , And Burton, P.A. S. , “Effects of peptide structures on transport in Caco-2 cells”, Pharm. Res. 8, 1453-1460, (1991))
[Non-Patent Document 2]
Abshowski and Davis ("Soluble polymer-enzyme adduct""Enzyme as a drug", Eds. Holchenbeg and Robert, J. Wiley and Sons, New York, NY (1981))
[Non-Patent Document 3]
In Bosch et al., Pharmacological Research Communications, 14, 11-120 (1982), others
[Non-Patent Document 4]
R. Igarashi et al., "Proceed. Inter. Symp. Control. Rel. Bioad. Materials, 17, 366 (1990).
[Non-Patent Document 5]
T. T. Taniguchi et al., Ibid., 19, 104, (1992)
[Non-Patent Document 6]
G. J. et al. Russell-Jones, ibid., 19, 102, (1992)
[Non-Patent Document 7]
M.M. Baudis et al., Ibid., 19, 210, (1992)
[Non-Patent Document 8]
M.M. Surfan et al., Science, 223, 1081, (1986)
[Non-patent document 9]
Y. W. “New Drug Delivery System” by Chain, 1992, NY, New York, Marcel Decker
[Non-Patent Document 10]
Santiago, N.C. Milestain, S. J. et al. Rivera, T. Garcia, E.C. Chang, T. C. Bowman, R.W. A. And Bach, D.C. "Immune immunization with influenza virus M protein (Ml) microspheres" Abstract No. A221, Inter. Symp. Control. Rel. Bioad. Materials, 19, 116 (1992) Minutes)
[Non-Patent Document 11]
Bantin et al. ("Pancreas Extract in the Treatment of Diabetic Martias", Can. Med. Assoc. J., 12, 141-146 (1992)).
[Non-Patent Document 12]
Bunlange J, Langgea L, Hebrand S, and Borland A, “Chemical Stability of Insulin, 1. Degradation during Storage of Pharmaceutical Formulations”, Pharm. Res. , 9, 715-726 (1992)
[Non-Patent Document 13]
Bunlange J, Hebrand S and Hogard P “Chemical stability of insulin, 2. Formulation of high molecular weight modified products during storage of pharmaceutical formulations” Pharm. Res. , 9, 727-734 (1992)
[Non-Patent Document 14]
Robins. D. C, Cooper. S. M, Fine Bark. S. E. And Mead P. M, "Antibodies to supply-bound aggregates of insulin in the blood of diabetics using insulin" Diabetes, 36, 838-841 (1987).
[Non-Patent Document 15]
MAYIOS. M, Mead. P. M, Gainer. D. H and Robins. D. C "The source of circulating aggregates in patients with type 1 diabetes is therapeutic insulin." J. et al. Clin. Invest, 77, 717-723 (1986)
[Non-Patent Document 16]
Ratner R.D. E, Philips. T. T. M and Steiner. M "Chronic skin allergy caused by high molecular weight insulin aggregates" Diabetes, 39, 728-733 (1990)
[0029]
[Problems to be solved by the invention]
An object of this invention is to provide the conjugate | zygote of the protein which can exhibit a biological effect, and a pharmaceutical composition containing the same.
[0030]
[Means for Solving the Problems]
The present invention relates generally to conjugate-stabilized (poly) peptide and protein compositions and formulations, and methods for making and using the same.
[0031]
More specifically, the present invention relates, in its broad composition form, to covalently linked peptide conjugates, in which the peptide incorporates a hydrophilic component such as, for example, a linear polyalkylene glycol. It is covalently bonded to one or more molecules of the polymer that it comprises as a component of, and the polymer contains a lipophilic component as its integral component.
[0032]
In one particular form, the present invention provides a physiologically active peptide comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the physiologically active peptide is covalently bonded to a polymer. With respect to a physiologically active peptide composition, the peptide, the linear polyalkylene glycol component and the lipophilic component are adapted to be compatible with each other, and the physiologically active peptide in the physiologically active peptide composition is physiologically active. As compared with the case where a chemically active peptide is used alone (that is, a non-conjugated form having no polymer attached thereto), the in vivo resistance to degradation of the enzyme can be improved.
[0033]
In another form, the present invention provides a three-dimensional consisting of a physiologically active peptide covalently linked to a polysorbate complex comprising (i) a linear polyalkylene glycol component and (ii) a lipophilic component. A physiologically active peptide composition having physical compatibility, wherein the physiologically active peptide, the linear polyalkylene glycol component and the lipophilic component are arranged to be compatible with respect to each other; (a) the lipophilic component Is available from the outside in a three-dimensional form, and (b) the physiologically active peptide in the physiologically active peptide composition is compared to the physiologically active peptide alone, In vivo resistance to degradation by enzymes is increased.
[0034]
In yet another aspect, the present invention relates to a multi-ligand conjugate peptide complex comprising a triglyceride body component, the complex comprising: That is, a biologically active peptide covalently bonded to the triglyceride body component through a polyalkylene glycol spacer moiety bonded at the carbon atom of the triglyceride body component, and directly to be covalently bonded to the carbon atom of the triglyceride body component At least one fatty acid component attached, or covalently bonded through a polyalkylene glycol spacer moiety.
[0035]
In such multi-ligand conjugated peptide conjugates, the α and β carbon atoms of the biologically active component of the triglyceride have a fatty acid component that is either directly covalently bonded or indirectly covalently bonded through a polyalkylene glycol spacer moiety. Alternatively, the fatty acid component is covalently attached to the α and α ′ carbons of the triglyceride body component directly or through a polyalkylene glycol spacer moiety, and the biological peptide is shared to the β carbon of the triglyceride body component. When attached, the triglyceride body component is covalently bonded directly or indirectly through a polyalkylene spacer component. It will be appreciated that a variety of structural, compositional and compatible forms are possible for a multiligand conjugated peptide consisting of a triglyceride body component within the scope of the above description.
[0036]
In such multi-ligand conjugated peptide complexes, it is advantageous to covalently attach the biologically active peptide to the triglyceride-modified body component through an alkyl spacer group, or alternatively other acceptable within the broad scope of the invention. It can also be attached through possible spacer groups. As used herein, acceptable spacer groups mean that they are stereochemical, compositional, and have acceptable properties specific to the final application.
[0037]
In yet another aspect, the present invention relates to a polysorbate complex comprising a polysorbate component comprising a triglyceride body covalently bonded to α, α ′ and β carbon atoms, the complex having a functional group comprising the following groups: That is, it has (i) a fatty acid group and (ii) a biologically active component covalently bonded, such as a physiologically active component covalently bonded to an appropriate functional group of, for example, a polyethylene glycol group Polyethylene glycol group.
[0038]
Such covalent bonds are covalently linked, for example, directly to the functional moiety of the hydroxy end of the polyethylene glycol group or indirectly, for example by reactively capping the hydroxy end of the polyethylene glycol group with a terminal carboxy functional moiety spacer group. The capped polyethylene glycol group that is attached and formed has a terminal carboxy functional moiety so that a physiologically active moiety can be covalently attached to this functional moiety.
[0039]
In yet another aspect, the present invention relates to a stable, water-soluble conjugated peptide complex comprising a physiologically active peptide covalently bound to a physiologically compatible polyethylene glycol modified glycolipid component. In such a complex, the physiologically active peptide can be covalently linked to a physiologically compatible polyethylene glycol-modified glycolipid component by covalent bonding at the free amino acid group of the peptide, where possible covalent The bonds can be separated in vivo by biochemical hydrolysis and / or protein hydrolysis. The physiologically compatible polyethylene glycol-modified glycolipid component advantageously comprises a polysorbate polymer such as, for example, a polysorbate polymer, which groups are monopalmitate, dipalmitate, monolorate, dilorate. , Trilorate, monoleate, dioleate, trioleate, monosterate, distyrate and tristyrate. In such a complex, the physiologically compatible glycolipid-modified glycolipid component suitably contains a polymer selected from a group consisting of a polyethylene glycol ether of a fatty acid and a polyethylene glycol ester of a fatty acid. Where it includes fatty acids such as fatty acids selected from the group consisting of rick, palmitic, auric and stearic acid. In the above complex, physiologically active peptides include, for example, insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic release factor, prolactin, thyroid stimulating hormone, endorphin, It can be formed with a peptide selected from the group consisting of enkephalin, vasopressin, non-naturally occurring opioids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin and papain.
[0040]
In another form, the present invention provides a stable, water-soluble conjugated insulin complex consisting of insulin or proinsulin covalently linked to a physiologically compatible polyethylene glycol modified glycolipid component, and a pharmaceutically acceptable The present invention relates to a dosage form for oral administration comprising a carrier for treating insulin deficiency.
[0041]
In yet another aspect, the present invention is a method of treating insulin deficiency in a human or non-human mammal subject deficient in insulin, wherein the insulin is covalently linked to a physiologically compatible polyethylene glycol modified glycolipid component. Or a method of treatment comprising the step of orally administering to a patient an effective amount of conjugated insulin comprising a stable, water-soluble conjugated insulin complex consisting of covalently bound proinsulin.
[0042]
As used herein, the term “peptide” is taken to have a broad meaning including a native polypeptide having a molecular weight of about 10,000 or less and a protein having a molecular weight of about 10,000 or more, where The molecular weight is the average molecular weight. As used herein, the term “covalently linked” refers to one or more intervening, such as bridges, spacers or one or more linking components, in which specific components are directly covalently bonded to each other. It is meant to be covalently bonded to each other indirectly through the components. The term “bonded together” shall mean that certain components are covalently bonded to each other or to each other, eg, by hydrogen bonding, ionic bonding, van der Waals forces, and the like.
[0043]
Thus, the present invention is directed to various therapeutic (in vivo) compositions, wherein the peptide component of the conjugated peptide complex is a physiologically active or biologically active peptide. It is. In a composition comprising such a peptide, conjugation of the peptide component to a polymer comprising hydrophilic and lipophilic components can be direct or indirect (through a suitable spacer group) and covalently linked together. It can be structurally disposed within the polymeric junction structure in any suitable manner, including direct or indirect covalent bonding. Thus, various peptide spacers can be adapted to a wide range of implementations of the invention, as required or desired for a given ultimate therapeutic purpose.
[0044]
In another form, the covalently bonded peptide composition as described above can utilize a peptide compound intended for diagnostic or in vitro application, in which case the peptide is, for example, a diagnostic reagent. Complementary to diagnostic conjugates for immunoassays or other diagnostic or in vitro applications. In such non-therapeutic applications, the peptide conjugates of the invention may be formulated in a stable composition with increased resistance to degradation, for example formulated in a compatible solvent or other solvent-based formulation. It can be used very effectively as a stabilized composition.
[0045]
For the therapeutic purposes described above or for non-therapeutic applications (eg, diagnostics), the present invention, in its broad form, relates to covalently conjugated peptide conjugates, in which case, for example, hydrophilic properties such as polyalkylene glycol moieties The peptide is covalently linked to one or more molecules of the polymer that comprise the component and, for example, a lipophilic component such as a fatty acid component as an integral part of the polymer. In one preferred form, the peptide is covalently linked to the peptide by one or more molecules of a linear polyalkylene glycol polymer whose integral part is a lipophilic component such as a fatty acid component. be able to.
[0046]
In another particular broad form, the present invention relates to non-covalently linked peptide conjugates, wherein the peptide is a hydrophilic component such as a polyalkylene glycol moiety and a parent such as a fatty acid moiety. Related in a non-covalent manner to one or more components of the polymer containing the oily component as an integral part thereof. The polymer can have various structures and arrangements similar to those described for the polymer in the covalently conjugated peptide complex described above, but the peptide is bound to the polymer molecule in a covalent manner. Rather, it is related to the polymer by methods such as, for example, related bonds, hydrogen bonds, ionic bonds or conjugation, van der Waals bonds, molecular encapsulation or association (specific peptides).
[0047]
Such non-covalent association of the peptide component and the polymer component utilizes the peptide component, eg, for therapeutic (eg, in vivo) applications, and also, eg, diagnostic or other (in vitro) applications. Therefore, peptide components other than those for treatment can be used.
[0048]
In the conjugated peptide composition thus related, the polymer component can be selected in a selective manner within the skill of the artisan (eg, to allow the polymer to bind hydrogen to each peptide). Appropriately configured, modified, or appropriately functionalized so as to provide an associable bonded body.
[0049]
Other aspects, features and applications of the invention will become more apparent from the following disclosure and claims.
[0050]
DETAILED DESCRIPTION OF THE INVENTION
Detailed Description of the Invention and Preferred Embodiments The modification of a peptide with a non-toxic, non-immune polymer results in several advantages. If the product (polymer-peptide conjugate) is modified in such a way that it retains all or most of its biological activity, the following properties are obtained: That is, the function of entering the epithelium of the living body is improved. The denatured peptide is protected from proteolytic digestion and subsequent loss of activity. Assimilation to the in-vivo transport system is increased. Chemical stability against acidity in the stomach is improved. The lipophilicity and hydrophilicity of the polymer are optimally balanced. Proteinaceous substances are effective as an alternative treatment method after oral or parenteral administration when given the improved properties described above. The modified peptides can also be used for administration through other routes such as the nose and skin.
[0051]
In non-therapeutic applications, peptide methods can be converted to polymers according to the methods of the present invention by diagnostic and / or reagent conjugate-stabilization of peptides containing end-use peptide precursors or intermediates or other products. The advantages associated with covalent bonding are obtained. This formed covalent peptide is resistant to environmental degradation factors including solvent or solution mediated degradation processes. As a result of increased resistance to degradation, the shelf life of the active peptide component can be significantly increased, which can increase the effectiveness of the composition containing the peptide for its particular end use application of the peptide. it can.
[0052]
As a result of covalently bonding the peptide to the polymer by the method of the present invention, degradation due to hydrolysis is effectively minimized, and stabilization in vitro and in vivo can be realized.
[0053]
Similar advantages are obtained when the therapeutic, diagnostic or reagent peptides are associated in non-covalent association and conjugated with the method of the present invention.
[0054]
As an example of an embodiment of the present invention, by using insulin covalently bonded to a polymer component, the time during which the polymer is decomposed from the peptide (insulin) due to the nature of the bond containing a degradable chemical covalent bond. Can be controlled. This degradation can be done by enzymes or chemical mechanisms. The conjugated polymer-peptide complex is active in nature. Full activity is obtained after enzymatic degradation of the polymer from the peptide. Furthermore, chemical denaturation allows, for example, attached peptides such as insulin to enter the cell membrane.
[0055]
In a preferred form of the invention, the property of increasing the membrane permeability of lipophilic fatty acid residues is incorporated into the body of the conjugate polymer. In this regard, when insulin is used as a peptide of interest, a derivative of a fatty acid polymer of insulin, Phe that similarly promotes absorption of insulin in the intestine. ^B1 And Lys ^B29 If the amino group is converted to a carbamyl group with a long-chain fatty acid polymer, a compound having a certain degree of hypoglycemic effect can be obtained. This derivatization increases the stability of insulin in the intestinal mucosa and also increases the absorbability from the small intestine.
[0056]
The above explanation mainly explains the use of insulin as a peptide compound in the various compositions and formulations of the present invention, but the usefulness of the present invention is not limited thereto, and the present invention is not limited thereto. It can also be applied to any peptide that can be covalently or associatively conjugated in the manner of: , Hypothalamic release factor, prolactin, thyroid stimulating hormone, endorphin, antibody, hemoglobin, soluble CD-4, coagulation factor, tissue plasminogen activator, enkephalin, vasopressin, non-naturally occurring opioid, superoxide dismutase, interface Ron, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, papain, alkaline phosphatase, and other suitable enzymes, hormones, proteins, polypeptides, enzyme protein conjugates, antibody hapten conjugates , Virus epitopes and the like, but not limited to these.
[0057]
One object of the present invention is to provide a polymer suitable for conjugation with peptides in order to obtain the desirable properties described above. Another purpose is to use the modified peptide to administer the peptide in vivo for a long time. Yet another object is to use techniques to orally administer the peptide in its active form.
[0058]
Yet another object is to employ relationally conjugated peptides for use in non-immunological assays, diagnostics and other therapeutic (eg, in vitro) applications. Yet another object of the present invention is to provide a peptide composition in a stable state, including a covalently bonded composition suitable for various in vivo and in vitro applications, and various in vivo and in vivo It is to provide related and conjugated peptide compositions in a non-covalent binding state suitable for other applications.
[0059]
Within the broad scope of the present invention, a single polymer molecule can be employed for conjugation with multiple peptides, and in a wide range of embodiments of the present invention, various conjugating agents for a given peptide can be used. It is advantageous to use a polymer, and a combination of such methods can be employed. Furthermore, the peptide composition joined in a stable state can be applied in both in vivo and in vitro applications. Further, it will be appreciated that the bonding polymer may utilize any other group, molecule or other bonding material as suitable for the final application. As an example, in certain applications, it may be useful to covalently bond a functional component to the polymer that imparts UV degradation resistance, or oxidation resistance or other properties or characteristics to the polymer. As yet another example, in some applications, it may be advantageous to functionalize the polymer to improve various properties or characteristics of the overall bonding material by having the same reactivity or cross-linking properties. It is. Thus, for the intended purpose, the polymer can include functionalities, repeat groups, linkages or other component structures that do not interfere with the effectiveness of the covalently bonded composition. Other objects and advantages of the present invention will become more apparent from the following disclosure and claims.
[0060]
An exemplary polymer that can be usefully employed to achieve these desirable characteristics is described below with respect to an exemplary reaction process. In covalently bonded peptide applications, the polymer is functionalized and then binds to the free amino acids of the peptide to form a labile conjugate that allows the labile bond to remain active. can do. Next, if the bond is broken by chemical hydrolysis and protein hydrolysis, the activity of the peptide is improved.
[0061]
Polymers utilized in the present invention include edible fatty acids (lipophilic ends), polyethylene glycol (water soluble ends), acceptable sugar components (ends that interact with receptors), and spacers for peptide attachment. Various molecular components can be included as appropriate. Of the polymers to be selected, polysorbate is particularly suitable, and in the following description, this polysorbate is selected to illustrate the various embodiments of the present invention.
[0062]
Of course, the scope of the present invention is not limited to polysorbates, and various other polymers containing the above-described components can be effectively employed within the broad scope of the present invention. In polymer structures, it may be desirable to remove one such component and retain the other without losing the purpose of the present invention. Where such measures are desired, the components that are preferably removed without losing the objects and advantages of the present invention are sugars and / or components.
[0063]
It is preferable to work with a polymer having a molecular weight in the range of 500 to 10,000 daltons.
[0064]
In practicing the present invention, it is advantageous to incorporate a polymer system directed to a C2-C4 alkyl polyalkylene glycol, preferably a polyalkylene glycol residue of polyethylene glycol (PEG).
[0065]
If such PEG residues are present, the polymer and the corresponding polymer peptide conjugate will be hydrophilic. Certain glycolipids that stabilize proteins and peptides are known. This stabilization mechanism involves associating the glycolipid fatty acid component with the hydrophobic region of the peptide or protein, thus preventing aggregation of the protein or peptide. Aggregated peptides are also known to be less absorbed by the small intestine than natural peptides. Thus, the present invention is directed to a polymer-peptide product in which a peptide such as, for example, insulin is conjugated to a hydrophilic or hydrophobic residue of the polymer. The peptide is provided with the fatty acid portion of the polymer so that it can be associated with a hydrophobic region, thereby preventing aggregation in solution. Because of this, the polymer-peptide conjugate formed is stable (against chemical and enzymatic hydrolysis) due to PEG residues and water soluble due to fatty acid-hydrophobic region interactions. There is no tendency to do.
[0066]
In the practice of the present invention, polyalkylene glycol derivatives have a number of advantageous properties when forming polymer-peptide conjugates when associated with the following polyalkylene glycol derivatives. That is, it has a high degree of biocompatibility that increases water solubility and does not exhibit antigenicity or immunological responsiveness. The polyalkylene glycol derivative does not biologically deteriorate in vivo. And excretion by biological organs is easy.
[0067]
The polymer employed in the practice of the present invention contains lipophilic and hydrophilic components, and the polymer-peptide conjugate formed is extremely effective in oral, parenteral and other physiological administration methods ( Bioactive) and extremely effective in applications other than living organisms.
[0068]
What is shown below is an example of the polymer-peptide conjugate of the present invention. For convenience of reference, the conjugate 1, the conjugate 2, the conjugate by covalent bond shown as the conjugate 3, etc. Where “ins” is the specific value of insulin, m, n, w, x, y as described below.
[0069]
The joined body 1 is represented by the following formula.
Embedded image

Wherein w + x + y + z = 20 and R is oleic acid: (CH _Three (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ C (O)-. )
The joined body 2 is represented by the following formula.
Embedded image

The joined body 3 is represented by the following formula.
Embedded image

[0070]
Conjugate 1 features a commercially available polysorbate monooleate at the center of the polymer system, ie a sugar derivative used in many pharmaceutical fields. While the lipophilic and absorbency enhancing properties are conferred by the oleic acid chain, the polyethylene glycol (PEG) residue provides a hydrophilic (hydrogen bond promoting) environment. Insulin is attached through a carbonate linkage adjacent to the PEG region of the polymer.
[0071]
In conjugate 2, the sugar residue is removed, but insulin is again attached to the polymer through the carbonate conjugate adjacent to the hydrophilic PEG region of the polymer. The lipophilic fatty acid region of the polymer is located some distance away from the point of attachment to insulin.
[0072]
The configuration described above with respect to the conjugate 2 is reversed for the conjugate 3. Again, the sugar residue is removed, but in this structure the lipophilic fatty acid residue is closest to the point of attachment to insulin and the affinity PEG region is also the point of attachment through the carbonate conjugate. It is in the position away from.
[0073]
Various alignments of the hydrophilic and lipophilic regions with respect to the point of attachment of the polymer to the peptide are possible in a wide range of implementations of the present invention, and such changes can be made to reduce the lipophilic and hydrophilic regions of insulin. A well-balanced polymer is obtained. In conjugates 1, 2 and 3, the point of attachment of the carbonate conjugate between the polymers is Gly. ^A1 It is preferable that it is an amine functional group. As described above, multiple displacement polymers can be attached to one of each molecule of the peptide. For example, when the second polymer is attached to insulin, the attachment point is Phe. ^B1 Preferably through the amine functional group. At least in theory, the third polymer is ^B29 It can be attached to the amine functional group. However, it has been empirically confirmed that attaching two polymers per insulin molecule is the maximum reasonable derivatization limit.
[0074]
In practicing the present invention, various methods for attaching polymers to peptides are available and will be described in further detail below. When dealing with proteins and polypeptides, it should be understood that the particular residue group within the peptide is important in terms of its overall biological integrity. It is important to select an appropriate binder that does not unduly interfere with such residues. In some cases, it is difficult to block binding and thus block the activity of these residues, but some activity is lost at the expense of increased stability while retaining the beneficial properties conferred. May be. For example, in in vivo applications, administration frequency can be reduced to reduce costs and increase patient convenience.
[0075]
In accordance with the present invention, the polymer utilized for the protein / peptide conjugate is set to include good physical properties that allow it to achieve the desired purpose. The absorption enhancer allows the penetration of the peptide through the cell membrane, but does not improve the stability of the peptide. -Peptide conjugates can be utilized. Thus, one form of the invention relates to the inclusion of fatty acid derivatives in the polymer to minimize penetration enhancers.
[0076]
In the covalently bonded polymer-peptide conjugate of the present invention, the peptide can be covalently attached to the water-soluble polymer by an unstable chemical bond. This covalent bond of peptides and polymers can be broken down by chemical or enzymatic reactions. This polymer-peptide polymer retains an acceptable degree of activity. When the polymer is completely degraded from the peptide, full activity of the peptide as a component is realized. At the same time, a portion of the polyethylene glycol is present in the conjugated polymer, giving the polymer-peptide a high degree of water solubility and long-term blood circulation function. Glycolipids are usually associated with polymers and their fatty acid components occupy the hydrophobic region of the peptide, thereby preventing aggregation. Aggregation of peptides results in insufficient absorption in the small intestine. Peptides that do not aggregate are more easily absorbed by the small intestine. For this reason, the inclusion of glycolipids in the binding polymer serves to increase stability and prevent peptide aggregation after conjugation. The above-described denaturation imparts improved meltability, stability and thin film affinity properties for the peptides. As a result of these improved properties, the present invention allows parenteral and oral administration of both active polymer-peptides, and in biological applications, after degradation by hydrolysis, the peptides themselves Realize usability.
[0077]
The polymers used in the examples described below can be classified into glycolipids modified with polyethylene glycol and fatty acids modified with polyethylene glycol. Among suitable bonding polymers, mention may be made of polysorbates comprising monopalmitate, dipalmitate, tripalmitate, monolorate, dilorate, trialoleate, monooleate, trioleate, trioleate, monostyrate, distyrate and tristyrate. The average molecular weight of the polymer obtained from each combination is preferably in the range of about 500 to about 10,000 daltons. Alternative polymers suitable for this example are polyethylene glycol ethers or esters, such fatty acids are loric, palmitic and stearic acid, and the average molecular weight of the polymer is between 500 and 10,000 daltons Range. The polymer preferably has a derivative group, in which case such a group can end up with polyethylene glycol or a fatty acid. This derivatizing group can also be located in the polymer and thus can function as a spacer group between the peptide and the polymer.
[0078]
Several methods for modifying the fatty acid sorbitan to obtain the desired polymer are described in more detail below with reference to molecular structures. Polysorbate is an ester of sorbitol and its anhydride, which is copolymerized with about 20 ethylene oxide molecules for each sorbitol and sorbitol anhydride molecule. Below, the structure of a typical polymer is shown.
[0079]
Embedded image

[0080]
The sum of w, x, y, z is 20 and R ₁ , R ₂ And R _Three Are each independently selected from the group consisting of loric, oleic, palmitic and stearic acid groups, or R ₁ , R ₂ Are each hydroxyl, while R _Three Is a loric, palmitic, oleic or stearic acid group. These polymers are commercially available and are used as drug formulations. If a high molecular weight polymer is desired, the polymer is synthesized from a glycolipid such as sorbitan monolorate, sorbitan monooleate, sorbitan monopalmitate or sorbitan monostearate and a suitable polyethylene glycol. be able to. The structure of glycolipid that can be used as a starting reagent is described below.
[0081]
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[0082]
When constructing a glycolipid polymer substituted with polyethylene glycol at three positions, the desired polyethylene glycol having two free hydroxyls at the terminal is used with 1 mole of trityl chloride to form a trityl group in polyzine. Protect with one terminal. The remaining free hydroxyl groups of the polyethylene glycol are replaced with tosylate or bromide. The desired glycolipid is melted in a suitable inert solvent and treated with sodium complex. Protected polyethylene glycol tosylate or bromide is melted in an inert solvent and added in excess to the glycolipid solution. The product is treated with a solution of paratoluenesulfonic acid in an anhydrous inert solution at room temperature and purified by column chromatography. The structure of this conversion will be described below.
[0083]
Embedded image

[0084]
By adjusting the molar equivalents of the reagents and using an appropriate molecular weight range of polyethylene glycol, mono- or di-substituted glycolipids having a desired range of molecular weight can be formed by the method described above.
[0085]
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[0086]
Here, n and m can be independently changed, and can be any value suitable for a specific peptide to be stabilized, for example, any one of 1 to 16.
[0087]
The sugar moiety of the glycolipid described above can be replaced with glycerol or aminoglycerol having the following structural molecular formula.
[0088]
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[0089]
In this modification, the primary alcohol is first etherified or esterified with a fatty acid component such as loric, oleic, palmitic or stearic and the amino group is derivatized with a fatty acid to give an amide or secondary as shown below. An amino group is formed.
[0090]
Embedded image

[0091]
Here, m can be any suitable value such as 10 to 16, for example.
The remaining first alcohol group is protected by a trityl group, while the second alcohol group is converted to the desired polymer by polyethylene glycol.
[0092]
Usually, polyethylene glycol has a group separating at one end and a metaoxy group at the other end. Polyethylene glycol is melted in an inert solvent and added to a solution containing glycolipid and sodium complex.
[0093]
This product is deprotected at room temperature in para-toluenesulfonic acid so that the desired polymer as shown below is obtained.
[0094]
Embedded image

[0095]
For example, including a fatty acid derivative in another part of the polyethylene glycol chain to obtain specific physicochemical properties similar to polysorbates substituted with two or three molecules of fatty acids such as polysorbate trioleate It may be desirable.
[0096]
The structure showing this polymer is shown as an open chain of polysorbate in the following reaction formula.
[0097]
Embedded image

[0098]
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[0099]
When synthesizing Polymer A, it is desirable to protect the hydroxyl component at the first and second carbons of glycerol, such as, for example, solketal. The remaining hydroxyl groups are converted to the sodium salt in an inert solvent and reacted with a halogenated or tosylated polyethylene glycol to be protected as an ester from one end of the polyethylene glycol. This glycerol protection is removed and the two free hydroxyl groups formed are converted to the corresponding sodium salts. These salts are reacted in an inert solvent with polyethylene glycol partially derived from fatty acids. The reaction takes place after the free hydroxyl has been converted to tosyl or bromine.
[0100]
Polymer G is synthesized in the same way, but first reacts an ester of a fatty acid halogenated at the terminal carbon of the acid with a protected glycerol.
[0101]
When synthesizing polymer C, it is desirable to start with 1,3-dihalo-2-propanol. The dihalo compound is melted in an inert solvent and treated with 2 moles of sodium salt of polyethylene glycol previously derived with 1 mole of fatty acid component. The product is purified by chromatography or dialysis. The dry product formed is treated with sodium hydride in an inert solvent. The sodium salt thus formed is reacted with a partially protected polyethylene glycol halo derivative.
[0102]
It may be desirable to omit the sugar portion of the polymer. The formed polymer still contains a polyethylene glycol component. The thin film affinity properties of the fatty acid component can be retained by replacing the inherent fatty acid with a lipophilic long fiber alkyne, thus maintaining biocompatibility. In one example of this example, polyethylene glycol having two terminal free hydroxyl groups is treated with sodium hydride in an inert solvent. One equivalent weight of the first bromine derivative of the fatty acid-like component is added to the mixture of polyethylene glycol solvents. The desired product is extracted in an inert solvent and, if necessary, purified by column chromatography.
[0103]
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[0104]
Where it is desired to form an ester linkage between the fatty acid and polyethylene glycol, the acid acid chloride is treated with excess desired polyethylene glycol in a suitable inert solvent. The polymer is extracted in an inert solvent and further purified by chromatography if necessary.
[0105]
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[0106]
In certain examples of peptide modifications, it may be desirable to conjugate the fatty acid component directly to the peptide. In this case, the polymer is synthesized with an inducible functional group placed on the fatty acid molecule. A solution of the appropriate molecular weight monometaoxyl polyethylene glycol in an inert solvent is treated with sodium hydride, followed by addition of a solution containing an ethyl ester of a fatty acid having a separating group at the terminal carbon of the acid. The product is purified after solvent extraction and, if necessary, purified by column chromatography.
[0107]
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[0108]
Unprotect the ester by treatment with dilute acid or base.
[0109]
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[0110]
If it is desired to form a carbonate conjugate with the polypeptide, the carboxyl or ester is converted to a hydroxyl group by chemical reduction methods known in the art.
[0111]
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[0112]
The functional group used in the polypeptide conjugate is usually at the end of the polymer, but in some cases it may be preferred that the functional group be located in the polymer. In this situation, the guiding group functions as a spacer. In one example of this example, the fatty acid component can be brominated to a carboxyl group at carbon α and the acid component can be esterified. The experimental method for this type of compound is similar to that described above, and the product shown below is obtained.
[0113]
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[0114]
When long spacers are desired, the polyethylene glycol monoether can be converted to amino groups and treated with silicic anhydride derived from the fatty acid component. The primary amine that supports the desired polyethylene glycol is melted in a pH 8.8 sodium phosphorus buffer and treated with a substituted fatty acid component of silicic anhydride as shown in the equation below. The product is separated by solvent extraction and, if necessary, purified by column chromatography.
[0115]
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[0116]
The reaction process described above is provided for convenience of illustration only and can be used advantageously to denature peptides, such as meltability, stability, etc., in the practice of the broad scope of the invention. It should not be construed to be limited to reactants and structures that can be utilized to achieve affinity with cell membranes for parenteral and oral administration.
[0117]
The present invention provides biologically compatible polymer conjugates with biologically active long molecules, diagnostic reagents such as peptides, proteins, enzymes, growth hormones, growth factors and the like. . Such long molecule compounds can be formed with alpha amino acids joined within a single amide linkage to form peptide oligomers and polymers. Depending on the function of these substances, the peptide component can be a protein, enzyme, growth hormone or the like. For clarity, these substances are collectively referred to below as peptides and denoted Pr. In all cases, biologically active peptides have a free amino or carboxyl group, and the linkage of the polymer and peptide is generally through the free amino or carboxyl group.
[0118]
The peptides selected for the description of the invention are particularly useful in the fields of medicine, agriculture, science and household and industry. These peptides are used in various fields such as enzymes utilized in replacement therapy, animal growth-promoting hormones, cell growth hormones in cell culture, or biotechnology and biological and medical diagnostics, for example. It can be an active proteinaceous substance. Enzymes include superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, papain, and peptide hormones include insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, There are parathyroid hormone, erythropoietin, hypothalamic release factor, prolactin, thyroid stimulating hormone, endorphin, enkephalin, vasopressin.
[0119]
The reaction of the polymer with the peptide to obtain a covalently bonded product is easily performed. For simplicity of explanation, the polymer is indicated by (P). If the polymer contains hydroxyl groups, the polymer is first converted to an active carbonate derivative such as para-nitrofelcarbonate. This active derivative then reacts with the peptide in a short time under mild conditions to generate a carbamate derivative that retains biological activity.
[0120]
Embedded image

[0121]
The reactions and reagents described above are for illustration only and are not limiting. Other active reagents that produce urethane or other conjugates can also be employed. Hydroxyl groups can also be converted to amino groups using reagents known in the art. Subsequent conjugation to the peptide through its carboxyl group results in the formation of an amide.
[0122]
When the polymer contains a carboxyl group, the polymer can be converted to a mixed anhydride and reacted with the amino group of the peptide to form a conjugate containing an amide conjugate. In other methods, the carboxyl group can be treated with a water soluble carbodiimide and reacted with the peptide to produce a conjugate comprising an amide conjugate.
[0123]
The activity and stability of the peptide conjugate can be changed by various methods such as changing the molecular weight ratio between the polymer and the peptide and using a polymer having a different molecular size. The meltability of the joined body can be changed by changing the ratio and size of the polyethylene glycol component contained in the polymer composition. The hydrophilic and lipophilic properties can be balanced by carefully combining the fatty acid component and the polyethylene glycol component.
[0124]
The following are some examples of denaturation reactions of the polymers of the present invention with peptide conjugates.
[0125]
Embedded image

[0126]
In the above reaction process involving I, J and K, a procedure has been clarified to modify the hydrophilic / lipophilic balance of the conjugated polymer. The ester group in the bonding polymer is easily subjected to hydrolysis by esterase. Therefore, the bonding polymer containing the ester group can convert the ester group into an ether group having high resistance to hydrolysis. The reaction process including L and M shows a state in which a hydroxyl group is converted to a carboxyl group. In this regard, the carboxyl group provides a carboxylate anion that has a better stabilizing function (forms an ion-harmonized complex) than hydroxyl, and this anion is not formed from such a complex. Other suitable anion source functional groups for forming coordinated ionic complexes comprising the polymers of the present invention include sulfuric acid and phosphate groups.
[0127]
In general, various techniques can be advantageously employed to improve the stability characteristics of the polymer-peptide conjugate of the present invention. These techniques include the following: That is, for example, a polymer such as the above-mentioned example in which an ester group is converted to another group is converted to a functional group with a group having excellent resistance to hydrolysis, and the polymer is joined so as to be suitable for a peptide stabilized by the polymer. To modify the lipophilic / hydrophilic balance of the polymer and to adjust the molecular weight of the polymer to an appropriate level relative to the molecular weight of the peptide stabilized by the polymer.
[0128]
A unique property of polyacrylate glycol-derived polymers that is valuable for applying the present invention for therapeutic purposes is that they are biocompatible or physiologically compatible overall. The polymer has various water-soluble properties and is not toxic. These polymers are non-antigenic and non-immunogenic and do not interfere with the biological activity of the enzyme. These polymers circulate for a long time in blood and are easily excreted from living organs.
[0129]
The products of the present invention have been found to be useful in preserving the biological activity of the peptide, eg, by administering it in water or an acceptable liquid medium, for example therapeutic administration. Can also be formulated for. It is administered parenterally or orally. Fine colloidal suspensions can be formulated for parenteral administration, provide a retention effect, or be administered orally.
[0130]
In the freeze-dried state, the peptide-polymer conjugate of the present invention is excellent in storage stability, and the solution formulation of the conjugate of the present invention is also excellent in storage stability.
[0131]
The therapeutic polymer-peptide conjugates of the present invention can be utilized for the prevention or treatment of any condition or condition for which the peptide component is effective.
[0132]
Furthermore, the polymer-peptide conjugates of the present invention can be used for the purposes of diagnosis of biological systems or species components, conditions or disease states and non-physiological systems.
[0133]
Furthermore, the polymer-peptide conjugate of the present invention can be applied to plant-based prevention or treatment of symptoms or disease states. As an example, the peptide component of the conjugate can be used in various plant systems and has insecticidal, herbicidal, fungicidal and / or insecticidal effects.
[0134]
Furthermore, the peptide component of the conjugate of the invention may be an antibody or may have antigenic properties for diagnostic, immunoassay and / or analytical purposes.
[0135]
When used for therapeutic purposes, the present invention may be applied to a method of treating an animal that has, or may be affected by, such a condition or condition, and is in need of such a condition. Or administering to the animal an effective amount of a polymer-peptide conjugate of the invention that is effective in the treatment of the condition.
[0136]
Subjects to be treated with the polymer-peptide conjugates of the present invention include both humans and non-human animals (eg, birds, dogs, cats, cows, horses), preferably mammals, and humans. Most preferably.
[0137]
Depending on the particular symptom or condition to be addressed, the animal is administered the polymer-peptide conjugate of the invention at any suitable therapeutically effective dose, which is known to those skilled in the art. It can be easily judged within the range and without unnecessary experimentation.
[0138]
In general, suitable dosages of the compound of formula 1 to achieve a therapeutic effect are in the range of 1 μg to 100 mg per kg of body weight per day and in the range of 10 μg to 500 mg per kg of body weight per day. Preferably, it is most preferably in the range of 10 μg to 50 mg per kg body weight per day. This desired dose is preferably administered in two, three, four, five, six or more doses at appropriate daily intervals. Such small doses can be administered in unit dosage form, for example, in the range of 10 μg to 1000 mg, and per unit dosage form, in the range of 50 μg to 500 mg of active ingredient. Preferably, it is most preferably in the range of 50 μg to 250 mg. Alternatively, if the recipient's condition requires, the dosage can be administered as a continuous infusion. Of course, the method of administration and dosage form are desirable and effective for a given therapeutic purpose. Affects therapeutic dose of compound.
[0139]
For example, the dose when administered orally is at least twice that of the same active ingredient when administered parenterally, typically 2 to 10 times, for example.
[0140]
The polymer-peptide conjugates of the present invention can be administered per se and in the form of pharmaceutically acceptable esters, salts and other physiologically effective derivatives. The present invention is also directed to drug formulations that are suitable for both veterinary and human treatment, the formulations comprising one or more polymer-peptide conjugates of the present invention as active agents.
[0141]
In such pharmaceutical and medical formulations, the active agent is utilized with one or more pharmaceutically acceptable carriers, and optionally with other therapeutic ingredients. The carrier must be pharmaceutically acceptable in that it is compatible with the other ingredients of the formulation and does not unduly harm the recipient. The active agent is administered in an amount that achieves the desired pharmacological effect as described above and in an amount that is suitable to achieve the desired daily administration.
[0142]
This formulation includes those suitable for parenteral and other modes of administration, including oral, rectal, buccal, topical, nasal, ocular, subcutaneous, intramuscular, There are intravenous, dermal, intrathecal, intraarticular, intraatrial, subarachnoid, bronchial, lymphatic sheath and intrauterine administration. A formulation suitable for oral and parenteral administration is preferred.
[0143]
When an active agent is utilized in a formulation that includes a liquid solution, the formulation is advantageously administered orally or parenterally. Where the active agent is employed in a liquid suspension formulation or as a powder in a biocompatible carrier formulation, it is advantageous to administer the formulation through the oral, rectal or bronchial.
[0144]
When the active agent is utilized directly in the form of a powdered solid, it is advantageous to administer the active agent orally. Alternatively, the bronchus may be administered by spraying the powder into a carrier gas to disperse the powder in a gaseous form and allowing the patient to aspirate the gas from a breathing circuit with an appropriate nebulizer device. .
[0145]
Formulations containing the active agents of the present invention can be conveniently provided in unit dosage form, and may be formulated by any method well known in the pharmaceutical arts. Such methods generally include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. Formulations are typically formulated by contacting the active compound uniformly and intimately with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, the shape of the product as desired. It is in the form of formulation administration.
[0146]
Formulations of the present invention suitable for oral administration can be provided as separate units such as capsules, cachets, tablets or troches, each of which is a powder or particle or aqueous solution, or a syrup, elixir, emulsion Or a predetermined amount of the active ingredient as a suspension in a liquid other than water, such as a donut.
[0147]
Tablets may be formed by compression or molding, optionally with the addition of one or more auxiliary additives. Compressed tablets may be compressed with a suitable machine so that the active compound is in a free flowing form like powders or particles, optionally with a binder, disintegrant, leveling agent, inert diluent, surfactant or Mix with release agent. Molded tablets comprising a mixture of the powdered active compound and a suitable carrier can be produced by molding in a suitable machine.
[0148]
For example, a syrup can be produced by adding an active compound to a concentrated aqueous solution of sugar such as sucrose and adding any auxiliary ingredients. Such auxiliary ingredients may also include flavoring agents, suitable preservatives, sugar crystallization retarders, and other component melt accelerators such as polyhydroxyl alcohols such as glycerol or sorbitol.
[0149]
Formulations suitable for parenteral administration include preparing a sterile aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient (eg, physiological saline solution). . Such formulations can include suspending and concentrating agents, or blood components, or other particulate systems that allow the compound to be targeted to one or more organs. Such formulations can also be provided in unit dosage or multiple dosage forms.
[0150]
Nasal sprayer formulations include aqueous solutions in which the active compound is purified as a preservative and isotonic agent. Such a formulation is preferably adjusted to a pH and isotonic state compatible with the nasal mucosa.
[0151]
Formulations for enteral administration may be provided as a suppository with a suitable carrier such as cocoa butter, hydrated fat or hydrated fat carboxylic acid.
[0152]
Ophthalmic formulations can be formulated in a similar manner of nasal spray, except that it is desirable to adjust the pH and isometric factors to suit the condition of the eye.
[0153]
Topical formulations consist of active compounds melted or suspended in one or more media, such as mineral oil, petroleum, polyhydroxyl alcohol, or other groups used in topical drug formulations. Including.
[0154]
In addition to the above components, the formulation of the present invention was selected from diluents, buffers, flavors, disintegrants, surfactants, thickeners, smoothing agents, preservatives (including oxidative preservatives), etc. One or more auxiliary ingredients may further be included.
[0155]
In non-therapeutic applications of the present invention, the polymer-peptide conjugate can utilize a covalent bond between the peptide and the polymer component, or alternatively, a non-covalent bond relationship. In addition, peptides and polymer components related when administering a therapeutic peptide agent by a suitable administration method as described below in connection with the description of the covalently bonded polymer-peptide conjugates of the present invention. Can be used.
[0156]
In such non-therapeutic applications, the associated peptide-polymer components, i.e., the peptide and polymer components, can be first formulated together to improve stability and resistance to degradation. Alternatively, these components can be, for example, multiple separate components that are mixed at the point of use and the polymer and peptide are associated in a mixture in which the component is formed. When not doing so, it will quickly deteriorate or undergo other deterioration effects. Regardless of the form of the related peptide and polymer composition, the present invention improves any property or form of the peptide or increases the effectiveness of the peptide with respect to the components of the peptide in the absence of such a related polymer. For relationships.
[0157]
Accordingly, an object of the present invention is to provide a suitable polymer that stabilizes a peptide in a solution in vitro as a suitable example of application other than treatment. For example, polymers can be employed to increase the thermal stability of peptides and increase resistance to degradation by enzymes. Promoting the thermostable properties of peptides via covalent bonds according to the method of the present invention results in improved shelf life, room temperature stability, diagnostic and research reagents, and robustness of kits such as, for example, immunoassay kits Means are obtained. As a specific example, in accordance with the present invention, such as when alkaline phosphatase is covalently or relatably bound to a suitable polymer and used as a reagent in a kit for colorimetric detection of an antibody or antigen in a biological fluid. Stability can also be imparted to phosphatase.
[0158]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. The following examples do not limit the invention.
[0159]
[Experimental Example I]
[Joint 1]
Polysorbate trioleate p-nitrophenyl carbonate To a solution of p-nitrophenyl chloroformate (0.8 g, 4 mol) in 50 ml of anhydrous acetonitrile is added dry polysorbate trioleate (7 g, 4 mol), then To the mixture is added dimethylraminopyridine (0.5 g, 4 mol). The reaction mixture is stirred for 24 hours at room temperature. The solvent is removed under low pressure and the precipitate formed is diluted with dry benzene and filtered through celite. The residue is refrigerated overnight in dry benzene and further precipitate is removed by filtration. Solvent is removed under low pressure and residual benzene is removed by venting at low pressure so that 6.4 g of polysorbate trioleate p-nitrophenyl carbonate is obtained.
[0160]
Binding of insulin to active polymer Bovine insulin solution (50 mg in phosphate buffer of 0.1 Mp, H8.8 in a solution of active polysorbate triolate p-nitrophenyl chloroformate (1 g) in distilled water. ) Is added. The pH is maintained by adding 1N NaOH as needed. The reaction mixture is stirred at room temperature for 2.5 hours. At this point, the mixture is subjected to gel filtration chromatography using Sephadex G-75. The conjugate 1 can be obtained by purifying by eluting with a phosphate buffer of 0.1 Mp, H7.0, and collecting the components with an automatic component collector. The polymer component is measured by trinitrobenzene sulfonic acid (TNBS) analysis, and the protein concentration is measured by the burette method. The molar ratio of polymer to insulin is measured as 1: 1.
[0161]
[Experimental example II]
[Joint body 2]
The terminal hydroxyl group of polyethylene glycol monostearate is activated by reacting with p-nitrophenyl chloroformate as described above. To a solution of active polymer (1 g) in distilled water is added bovine insulin (80 mg) melted in 0.1 M pH phosphate buffer at pH 8.8.
[0162]
This pH is maintained by careful adjustment with 1N NaOH. After stirring for 3 hours, the reaction mixture is quenched with excess glycine and subjected to gel filtration chromatography using Sephadex G-75. Insulin-polymer conjugates were collected and lyophilized. The protein component is measured by a burette analysis method to obtain a quantitative value.
[0163]
[Experimental Example III]
[Joint body 3]
To a solution of 12-bromo-1-dodecanol (1 mol) in dichloromethane containing tetrahydro-2- (12-bromododecanoxy) -2H pyrone pyridinium p-toluenesulfonate (P-TSA), dihydropyran (2 mol) Add. The reaction mixture is stirred for 24 hours, then washed twice with water and dried over anhydrous MgSO4. Dichloromethane is removed under low pressure. If necessary, the product formed is purified by chromatography on silica gel.
[0164]
Binding of Polyethylene Glycol with Tetrahydropyran Derivative The above-mentioned tetrahydropyran derivative melted in dry benzene is added to a solution of polyethylene glycol (1 mol) in dry benzene containing NaH (1 mol). The reaction mixture is stirred at room temperature for 24 hours. After that time, the mixture is eluted through a silica gel column with benzene. If necessary, further purification is performed by column chromatography. The protected tetrahydropyran group is removed by treatment with p-TSA at room temperature. If necessary, purify the final product by column chromatography. The hydroxyl group of the polymer is activated by reaction with p-nitrophenyl chloroformate as described above. Joining with insulin is performed as described for the joined body 1.
[0165]
[Experimental example IV]
A comparative study was conducted using bovine insulin on the polymer-insulin conjugate and natural insulin to determine its relative stability and activity in animals. When studying animals, the effect of polymer-insulin on blood pressure reduction was compared with that of natural insulin. Female and male white pupae with an average weight of 25 g were fasted overnight, and 5 were used as a group for each treatment performed at various stages for 2 days.
[0166]
Natural insulin by each test animal (group 1, 100 μg / kg, subcutaneous injection), natural insulin (group 2, 1.5 mg / kg, gavage).
[0167]
Conjugate 1) Group 3, 100 μg / kg, oral administration) or Conjugate 1 (group 4, 100 μg / kg, subcutaneous injection) was administered once at time zero. The additional group (Group 5) was not administered any kind of insulin and was loaded with Glycose 30 30 minutes before the predetermined collection time. Animals were fasted before treatment, day and night, and throughout the study.
[0168]
Test materials were formulated in phosphate buffered saline pH 7.4. After treatment with insulin, animals were loaded with a large glucose load (5 g / kg as a 50% solution) 30 minutes before the predetermined collection times of 0.5, 1, 2, 4, 8 and 24 hours. Oral administration). Each animal was given only one dose of insulin or conjugate 1 and one glucose load. At a predetermined collection time, blood was collected from the tail blood vessel, and blood glucose level was immediately analyzed using a one-touch digital glucose meter (life scan). The test results are listed in FIG. 1 for groups 1-5.
[0169]
The blood glucose level of Group 1 animals (natural insulin, subcutaneous injection) was approximately 30% of control (Group 5, untreated) animals at the 30 minute test time point. This hypoglycemic effect lasted only 3.5 hours in Group 1 animals. Orally administered natural insulin (Group 2) reduces blood glucose levels to a maximum of 60% of controls, and this maximum response occurs at 30 minutes after treatment with insulin. On the other hand, blood glucose levels in Group 3 animals (zygote 1, 100 μg / kg, po) decreased with the apparent delayed onset of hypoglycemic activity. The hypoglycemic activity of the Group 3 animals was more prominent than the Group 2 animals, even though the insulin dose administered to Group 3 was only 1/15 that was administered to Group 2. At all time points after 3 hours, the blood glucose levels of the animals in group 3 are lower than in the other treatment groups, with the greatest difference occurring at the 4-8 hour collection time point. The blood glucose levels of Group 4 animals (Conjugate 1, 100 μg / kg, sc) were the same course as those of Group 1 animals for the first 4 hours of the study. After 4 hours, the blood glucose level of group 2 is higher than that of the control (untreated, group 5), while at 8 hours, the blood glucose level of group 4 falls below 62% of the blood glucose level of group 5. , Kept below the blood glucose level of group 5.
[0170]
[Experiment V]
Using the non-joined form of insulin and the joined body 1 as test materials, the effect of insulin on male and female white pupae was tested. One purpose of this experiment is to determine whether or not there is an effect on blood glucose levels in the same manner as insulin when insulin in the form of conjugate 1 is injected subcutaneously. The second purpose is to determine whether or not there is an action of lowering blood glucose level when the insulin complex of conjugate 1 is orally administered, unlike free insulin. The results are listed in FIG. 2, where “insulin complex” means zygote 1.
[0171]
A reference blood sample was collected from 10 fast-treated and untreated Shirako moths (5 males and 5 females) for plasma glucose analysis. In FIG. 2, the reference value is indicated by a symbol “O”. Three additional groups (5 males and 5 females) were fasted overnight, and glucose was administered only by oral administration with Gabersch (5 g / kg body weight). At 30, 60 and 120 minutes after administration, blood samples were collected from 10 animals three times and analyzed for glucose. Both oral (po) and parenteral (sc) for fasted pupae groups (5 males and 5 females killed at each of 3 time points and blood analyzed) Commercial insulin and conjugate 1 were administered for each administration so that different treatment groups were obtained. The symbols shown for this treatment, administration route, and experimental results of FIG. 2 are as follows.
[0172]
(I) glucose (5 g / kg, po), symbol “●”, (ii) insulin (100 μg / kg, sc) and glucose (5 g / kg, po), symbol “▽” ”, (Iii) insulin (1.5 mg / kg, po) and glucose (5 g / kg, po), symbol“ ▽ ”, (iv) conjugate 1 (100 μg / kg, sc) .) And glucose (5 g / kg, po), symbol “□”, (v) Conjugate 1 (250 μg / kg, sc) and glucose (5 g / kg, po), symbol “□”, (vi) Conjugate (1.5 mg / kg, sc) and glucose (5 g / kg, po), symbol “Δ”. In these tests of Conjugate 1, the protein concentration in the administered solution is 0.1 mg protein / ml solution, and for comparison purposes, the protein concentration in the administered solution of 0.78 mg protein / ml solution. Insulin-polymer conjugates with some modified covalent bonds are included. (Vii) Modified conjugate 1 (100 pg / kg, sc) and glucose (5 g / kg, po), symbol “Δ”.
[0173]
Insulin was administered at 15 minutes prior to applying the glucose load. Glucose was orally administered by gavage (10 mg / kg of a 50% w / v solution in standard saline) to all groups except the reference animal group at a dose of 5 g / kg. When insulin was administered orally by gavage, the dose was 1.5 mg / kg (18.85 ml / kg of a 0.008% w / v solution in standard saline). When insulin was administered by subcutaneous injection, the dose was 100 μg / kg (2.5 μl / kg of a 0.004% w / v solution in standard saline).
[0174]
When the conjugate 1 polymer-insulin complex was orally administered by gavage, the dose was administered at a dose of 1.56 mg / kg (2.0 ml / kg of undiluted test material). When the polymer-insulin complex of Conjugate 1 was administered by subcutaneous injection, the dose was 100 μg / kg (1.28 μl / kg of the administered 0.78 mg / ml solution diluted 1:10). Or 250 μg / kg (3.20 ml / kg diluted 1 to 10 of the administered solution). Denatured conjugate pair 1 contained 0.1 ml / kg insulin / ml and was administered in an amount of 1.0 ml / kg to give a dose of 100 μg / kg.
[0175]
Glucose was measured using Gemini centrifugal force analysis and a commercially available glucose reagent kit. Along with this analysis, enzyme analysis was performed based on the reaction between ATP catalyzed by hexokinase and glucose, and glucose-6-phosphate dehydration reaction was performed to obtain NAOH. Similar specimens were analyzed and the average value was determined. Some plasma samples required dilution (1 to 2 or 1 to 4) to determine the very high glucose concentration in a particular sample.
[0176]
After applying the glucose load, the average plasma glucose value became high at 30 minutes, decreased at 60 minutes, and below the reference value at 120 minutes. When commercially available insulin was administered by subcutaneous injection (100 μg / kg body weight, this was very effective in preventing an increase in blood glucose level). However, when insulin was orally administered (at a large dose of 1.5 mg / kg), there was no effect on the increase in blood glucose level. This was expected because insulin, protein, is easily hydrolyzed within the digestive pathway and is not completely absorbed into the bloodstream.
[0177]
When Conjugate 1 was injected subcutaneously at either a 100 or 250 μg / kg dose, the conjugate was extremely effective in limiting the increase in blood glucose levels after applying a glucose load. At both 30 minutes and 60 minutes, 100 μg / kg of Conjugate 1 was administered, and then the mean plasma blood glucose level was significantly lower than when 100 μg / kg of free insulin was administered. The mean plasma blood glucose level when zygote 1 was administered at 250 μg / kg was low and not remarkably at 30 minutes, but remarkably low at 60 minutes and returned to the reference value at 120 minutes. When both 100 μg / kg of free insulin and 100 μg / kg of conjugate 1 were administered, the blood glucose level at 120 minutes was below the reference value.
[0178]
When denatured conjugate 1 was administered at a dose of 100 μg / kg, the blood glucose level significantly decreased at 30 minutes.
[0179]
[Experimental example VI]
[Preparation of polysorbate monopalmitate para-nitrophenyl carbonate]
The polysorbate monopalmitate is first dried by azeotropy using dry benzene.
[0180]
To a solution of dry polymer (2 g, 2 mol) in 10 ml dry pyridine is added para-nitrophenyl chloroformate (0.6 g, 3 mol). The mixture is stirred at room temperature for 24 hours. The reaction mixture is cooled with ice, diluted with dry benzene and filtered using a filter. This procedure is repeated and finally the solvent is removed on a rotary evaporator. Residual solvent is removed with a vacuum suction device. The amount of product is 1.8 g.
[0181]
[Experiment VII]
[Preparation of polysorbate / monopalmitate conjugate with insulin]
According to the conjugation reaction procedure of Experimental Example 1 described above, when 1 g of polysorbate monopalmitate and 80 mg of insulin were used and the reaction product was separated by HPLC, insulin-polysorbate monopalmitate covalently bonded conjugate Is obtained.
[0182]
[Experiment VIII]
[Preparation of enzyme-polymer conjugate]
Using the same method as described for Conjugate 1 of Experimental Example 1, alkaline phosphatase (AP) was conjugated to the polymer, and either the polymer to protein ratio was either increased or decreased. Was determined using 140 moles of polymer / oxygen mole and 14 moles of polymer / enzyme mole. The number of polymer groups per molecule of conjugate AP is 30 and 5, respectively, for high and low ratios of polymer.
[0183]
In order to obtain approximately 5 groups / molecule of alkaline phosphatase, the following procedure was adopted. 4.1 mg (unsalted) was melted in 0.05M sodium bicarbonate. To this solution was added the active polymer (0.75 mg) in water / dimethyl-sulfoxide and the solution was stirred at room temperature for 3-12 hours. The reaction mixture formed was dialyzed against salt solution (0.3 N · NaCl) for 12 hours in a dialysis tube (MW cutoff 12,000-14,000), and the dialysis solution was 4-6 times. Exchanged. The same procedure was performed for the high ratio. The total protein concentration of the dialyzed material was measured by the burette method.
[0184]
[Measurement of activity and stability test]
A. Phosphatase analysis was performed according to the method of Voller et al., WHO, 53, 55 (1976). Add a microwell aliquot (50 μl) and mix with 200 μl of the base solution (4-nitrophenyl phosphate, 10 g / l, pH 9.3 in 20% ethanolamine buffer) for 45 hours at room temperature. Keep warm. The reaction was stopped with 50 μl of 3M NaOH. This absorptance was measured at 405 nm in a microplate reader.
[0185]
The activity of phosphatase was compared with natural enzyme under various conditions. Diluted solutions containing similar concentrations of alkaline phosphatase and alkaline phosphatase-polymer conjugate were stored at various temperatures. The enzyme activity was tested periodically. Two polymers tested at 5 ° C., 15 ° C., 35 ° C. and 55 ° C. were compared to a control alkali phosphate stored at 5 ° C.
[0186]
As can be seen from Table A, the initial enzyme activity of both polymers was about 3 times greater than the control. Both polymer-enzyme conjugates are more thermostable than natural enzymes, particularly in the case of conjugates characterized by a high polymer to enzyme ratio.
[0187]
[Table 1]

[0188]
[Experiment IX]
[Joint 1]
A. To an insulin solution (50 mg) in 0.05 M sodium bicarbonate buffer at pH 9.2 was added an active polymer solution (1 g) in water-dimethyl sulfoxide and stirred at room temperature for 3 hours. The pH of the mixture is maintained by careful adjustment with 1N NaOH. The reaction mixture is then dialyzed against 0.1 M pH 7.0 phosphate buffer. The purified product is lyophilized. The protein content (48 mg) is measured by the burette assay. The number of polymer chains attached to insulin is determined by TNBS assay and is in a ratio of 2 moles polymerization to 1 mole insulin.
[0200]
[Experiment XVI]
Polymer-insulin is evaluated in a diabetic (BB) epilepsy model in the following manner. BB 鼠 is a reliable model of human insulin-dependent diabetes.
[0201]
BB sputum dies within 2 days unless given daily transdermal (sc) insulin. During the 14-day study, sc insulin was stopped and the animals were polymerized by oral administration of low (100 μg / kg / day), medium (3 mg / kg / day), high (6 mg / kg / day) doses— Switched to insulin treatment. The results (average survival time) are listed in FIG. The rapid switching from sc to oral administration is a particularly harsh test of polymeric insulin. The results show that low dose animals do not receive enough insulin to survive, medium dose groups have a slightly longer survival time, and some animals in high dose groups It can be seen that the patient survived during the study period (14 days) receiving administration of the administered polymer insulin. The polymer insulin was gavaged in an unsuitable formulation. The results of this study also show that pm is significantly reduced compared to am blood glucose levels, and animals are better controlled when administered multiple times a day rather than in large doses. I knew I would get it. Daily administration of polymer-insulin to normal (non-diabetic) animals results in a marked decrease in am blood glucose levels.
[0205]
BEST MODES FOR CARRYING OUT THE INVENTION Polypeptide polymer compositions and formulations according to the presently preferred conjugate stabilization of the present invention relate to covalently bound peptide conjugates, wherein the peptide is as an integral part thereof, for example, It is covalently bonded to one or more molecules of a polymer containing a hydrophilic component such as a linear polyalkylene glycol, and the polymer contains a lipophilic component as an integral part thereof.
[0206]
A particularly preferred form of the invention relates to a physiologically active peptide composition comprising a physiologically active peptide covalently bound to a polymer, the polymer comprising: That is, (I) a linear polyalkylene glycol component and (II) a lipophilic component. Here, the peptide, the linear polyalkylene glycol component and the lipophilic component are arranged in harmony with each other, and the physiologically active peptide in the physiologically active peptide composition is the physiologically active peptide alone Compared to the case of (i.e., in the form of a conjugate in which no bound polymer is present), the body's resistance to degradation by the enzyme is increased.
[0207]
Furthermore, in a preferred form, the present invention relates to a three-dimensionally coordinated physiologically active peptide composition comprising a physiologically active peptide covalently linked to a polysorbate complex comprising: That is, (i) a linear polyalkylene glycol component and (ii) a lipophilic component, wherein the physiologically active peptide is such that the linear polyalkylene glycol component and the lipophilic component are arranged harmoniously with respect to each other. The lipophilic component is available from the outside in a three-dimensionally harmonized state, and (b) the physiologically active peptide in the physiologically active peptide composition is a physiologically active peptide alone Compared to the increase in resistance to degradation by enzymes.
[0208]
In yet another preferred form, the present invention comprises a triglyceride body component having a biologically active peptide covalently bonded to the triglyceride body component through a polyalkylene glycol spacer group bonded at the carbon atom to the triglyceride body component. With respect to multi-ligand conjugated peptide conjugates, the conjugate consists of at least one fatty acid component that is either covalently bonded directly to a carbon atom of the triglyceride body component or covalently bonded through a polyalkylene glycol spacer component. In such multi-ligand conjugated peptide complexes, the α and β carbon atoms of the triglyceride biologically active component may be directly covalently attached or include a fatty acid component indirectly covalently bonded through a polyalkylene glycol spacer component. it can. Alternatively, the fatty acid component can be covalently bonded directly or through the polyalkylene glycol spacer component to the alpha carbon of the triglyceride body component, and the biologically active peptide can be directly covalently bonded or through the polyalkylene spacer component. It is indirectly covalently bonded to the β carbon of the triglyceride component.
[0209]
INDUSTRIAL APPLICABILITY OF THE INVENTION The present invention is directed to the use of the conjugated stable peptide composition disclosed herein for the oral administration of drugs and the treatment of conditions where peptides are insufficient.
[Brief description of the drawings]
FIG. 1 is a graph showing plasma glucose levels in mg / dL as a function of time in minutes for administration in the form of insulin itself and in complex form.
FIG. 2 is a diagram showing the plasma glucose level in mg / dL as a function of the time (hours) at which insulin is administered in various forms.
FIG. 3 is a graph of chymotrypsin digestion of insulin and OT insulin as a function of time.
FIG. 4 is a graph showing plasma calcium levels in sputum as a function of time when calcitonin conjugate is orally administered to sputum.
FIG. 5 is a bar graph showing the amount of serum and calcium in sputum as a function of time when polymer calcitonin OT1-ct or OT2-ct polymer calcitonin conjugate is orally administered to sputum. .
FIG. 6 shows a bar graph of polymer-insulin effects in diabetic epilepsy.
FIG. 7 is a graph showing the effect of oral administration of 001-insulin in a Cynomolgus monkey as a change in blood glucose concentration over time.
FIG. 8 is a bar graph showing the administration effect over the elapsed time when 001-insulin was orally administered to a Cynomolgus monkey.

Claims (12)

A polymer peptide conjugate conjugated so that the peptide is stably conjugated to one or more non-naturally occurring polymer molecules, the polymer molecule comprising a lipophilic portion and a hydrophilic portion, wherein the polymer A polymer peptide conjugate wherein the molecule is selected from the group consisting of compounds represented by the following formulae:

(In Formula (1), m is 10-16, n is 5-120, and in Formula (2), m is 10-16, n1 is 4-119, n2 is 5-120, n3 is 5-120. And X is O or OCH ₂ CONH—.)   The polymer peptide conjugate according to claim 1, wherein the peptide is a physiologically active therapeutic peptide.   The polymer peptide conjugate according to claim 1, wherein the peptide is a diagnostic peptide used for determining a certain symptom or presence / absence of a certain component by reaction in vivo or in vitro.   The polymer peptide conjugate according to claim 1, wherein the peptide is active in plants or components thereof.   The peptide is a physiologically active peptide, and the physiologically active peptide is enhanced in vivo in the polymer peptide conjugate as compared to the physiologically active peptide alone. The polymer peptide conjugate according to claim 1, wherein conformation of the lipophilic part and the hydrophilic part is determined so as to have resistance to enzymatic degradation.   The polymer peptide conjugate according to claim 1, wherein the peptide has a molecular weight of 500 to 10,000 daltons.   The peptide is insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic release factor, prolactin, thyroid stimulating hormone, endorphin, enkephalin, vasopressin, non-naturally occurring opioid, superoxide The polymer peptide conjugate according to claim 1, wherein the polymer peptide conjugate is selected from the group consisting of dismutase, ribonuclease, and papain.   The polymer peptide conjugate according to claim 1, wherein the peptide is insulin.   The polymer peptide conjugate according to claim 1, wherein the peptide is calcitonin.   The use method for manufacture of the oral preparation for the insulin deficiency treatment of the conjugate | zygote in any one of Claims 1-8, Comprising: The said peptide is an insulin.   An oral preparation for the treatment of insulin deficiency, comprising the conjugate according to any one of claims 1 to 8, and a carrier or a diluent, wherein the peptide is insulin. A polymer-insulin peptide conjugate in which an insulin peptide is conjugated so that it is stably conjugated to one or more non-naturally occurring polymer molecules, the polymer molecule comprising a lipophilic portion and a hydrophilic portion. A polymer peptide conjugate wherein the polymer molecule is selected from the group consisting of compounds represented by the following formulae:

(In Formula (5), m is 10-16, n is 5-120, and in Formula (6), m is 10-16 independently, and n is 5-120. )

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